Use of tnks inhibitors for regeneration of cartilage

ABSTRACT

The present disclosure relates to a method of treating arthritis by targeting Tankyrase. The methods according to the present disclosure can be advantageously used for regeneration of cartilage tissue and for treating osteoarthritis by maximizing the matrix synthesis in cartilage by inhibition of Tankyrase and regulation of other proteins related therewith.

BACKGROUND OF THE INVENTION Sequence Listing

The Sequence Listing submitted in text format (.txt) filed on May 13,2020, named “SequenceListing.txt”, created on May 6, 2020 (16.7 KB), isincorporated herein by reference.

Field of the Invention

The present disclosure is related to the regeneration of cartilage.

Description of the Related Art

The conventional research related to osteoarthritis (OA) has beenfocused on the studies identifying the mechanism of degeneration ofarthritis. Accordingly, the main factors leading to the degenerationmechanism are well known. Existing treatment strategies also focus onslowing the progression of the disease by suppressing degenerationfactors, and these strategies cannot have the fundamental therapeuticeffect on regenerating cartilage.

Cartilage tissue is a tissue that gradually degrades when it begins tobe damaged by aging or injury. Degenerative arthritis is a disease thatis afflicted by 4.41 million people in Korea as of 2014, and the demandfor treatment is rapidly increasing. However, drugs used to treatdegenerative arthritis remain at pain relief levels such as hyaluronicacid and anti-inflammatory drugs. The treatments that induce fundamentalregeneration of cartilage have not yet been developed, and research isin its infancy.

Korean Patent Application Publication No. 2014-0144508 relates to acomposition for treating damaged cartilage by regeneration thereof anddiscloses a composition comprising granulocyte macrophage-colonystimulating factor; GM-CSF as an effective ingredient for treatingdamaged cartilage by regenerating cartilage.

Korean Patent Application Publication No. 2005-0012226 relates toregeneration of cartilage by use of TGF-beta and chondrocyte anddiscloses a method of treating osteoarthritis by treating the cells withmembers of TGF super family.

However, no such documents disclose in connection with the treatment ofcartilage regeneration targeting the factor disclosed herein. In orderto fundamentally regenerate cartilage that has undergone degeneration,molecular mechanisms that regulate cartilage regeneration factors areneeded to be identified, and the development of a treatment strategythrough regulation of the factors is required.

SUMMARY OF THE INVENTION

The present disclosure is to provide a method treating arthritis orrelated disease through the regeneration of cartilage tissue bymaximizing the ability of the chondrocytes to synthesize matrices byregulating Tankyrase-SOX9 pathway.

In one aspect of the present disclosure, a method of treating arthritisin a subject in need thereof comprising the step of administering to thesubject an effective amount of an inhibitor of Tankyrase; or a modifiedadult stem cell in which the expression of Tankyrase is suppressed orTankyrase gene is knocked out, wherein the inhibitor of Tankyrase or themodified adult stem cell stabilizes the Sox9 protein or increases theconcentration of the Sox9 protein by inhibiting the Tankyrase activitypromoting the degradation of Sox9 protein.

In one embodiment, the inhibitor of Tankyrase leads to chondrogenicdifferentiation of an adult stem cells leading to chondrogenicregeneration.

In other embodiment, the inhibitor of Tankyrase is an agent that bindsto a nicotinamide sub-domain or region of ARTD domain which is acatalytic domain of a Tankyrase protein, an agent that binds to anadenosine sub-domain of a Tankyrase protein or an agent that binds to anunidentified domain of a Tankyrase protein.

In other embodiment, the agent that binds to a nicotinamide sub-domainof ARTD domain is XAV939{3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}or MN-66 {2-[4-(1-methylethyl)phenyl]-4H-1-benzopyran-4-one}; the agentthat binds to an adenosine sub-domain of a Tankyrase protein is IWR-1[4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolynyl-benzamide],JW55{N-[4-[[[[tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl]methyl]amino]carbonyl]phenyl]-2-purancarboxamide},WIKI42-[3-[[4-(4-methoxyphenyl)-5-(4-pyridynyl)-4H-1,2,4-triazol-3-yl]thio]propyl]-1Hbenz[de]isoquinoline-1,3(2H)-dion,TC-E5001{3-(4-methoxyphenyl)-5-[[[4-(4-methoxyphenyl)-5-methyl-4H-1,2,4-triazol-3-yl]thio]methyl]-1,2,4-oxadiazolor G007-LK{(E)-4-(5-(2-(4-(2-chlorophenyl)-5-(5-(methylsulfonyl)pyridine-2-yl)-4H-1,2,4-triazol-3-yl)vinyl)-1,3,4-oxadiazole-2-yl)benzonitrile};and the agent that binds to an unidentified domain of a Tankyraseprotein is G244-LM {3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazynyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}, or AZ6102{rel-2-[4-[6-[(3R,5S)-3,5-dimethyl-1-piperazynyl]-4-methyl-3-pyridynyl]phenyl]-3,7-dihydro-7-methyl-4H-pyrrolo[2,3-d]pyrimidine-4-one},or isomers or derivative thereof.

In other embodiment, the inhibitor is a siRNA that suppresses theexpression of Tankyrase gene into a Tankyrase protein.

In other embodiment, the siRNA is a dsRNA consisting of RNAs of SEQ IDNO: X1 and SEQ ID NO:X2; or a dsRNA consisting of RNAs of SEQ ID NO: X3and SEQ ID NO:X4.

In other embodiment, the adult stem cell is autologous or allogenic.

In other aspect, there is provided a method of promoting thedifferentiation of an adult stem cell into a cartilage cell by treatingthe stem cell with an inhibitor of Tankyrase.

Advantageous Effects

Here it was found that Tankyrase is an upstream regulator of SOX9, whichis known as an important factor in the formation of cartilage matrix,and the inhibition of Tankyrase can lead to the cartilage regenerationin vivo and in vitro useful for OA therapies. Here it was also foundthat the cartilage regeneration is possible by promoting the cartilagematrix protein synthesis of chondrocytes present in cartilage tissuesand the differentiation of MSC into chondrocytes. Thus the mechanismidentified herein can be advantageously used for treating variousdisease such as OA which can benefit from the cartilage regeneration byinhibiting Tankyrase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 11 are the results of the identification of Tankyrase as aregulator of cartilage anabolic axis. (a) Heatmap of Pearson correlationcoefficients of transcript levels for cartilage matrix genes from 16 BXDmouse strains. (b) Factor loadings plot of 14 cartilage matrix genes incartilage of the 16 BXD mouse strains in terms of transcript abundance,with Tnks and Tnks2 added to the plot. Pearson's r and P value displayednext to Tnks or Tnks2 point represent correlation strength betweenFactor 1 and Tnks or Tnks2. (c) Correlation between Tnks or Tnks2 andCol2a1 or Acan mRNA levels in the 16 BXD mouse strains. (d) Knockdownefficiency of various Tnks and Tnks2 siRNAs in primary cultured mousechondrocytes (n=3). siTnks #2 and siTnks 2 #3 were used throughout thisstudy. e-g mRNA and protein levels of cartilage-specific matrix genes inmouse chondrocytes treated with (e) control or Tnks and Tnks2 siRNAs(n=4) or (f, g) drugs (n=7). Col6a5, Col6a6, and Col13a1 mRNAs wereundetected. (h) Gene set enrichment analysis (GSEA) ofcartilage-signature genes in mouse chondrocytes transfected with siTnksand siTnks2 compared to control siRNA. (i) GSEA of cartilage-signaturegenes in chondrocytes treated with tankyrase inhibitors compared tovehicle. Cartilage-signature genes are listed in Table 8. Genesupregulated in mouse chondrocytes compared to mouse embryonicfibroblasts were selected as cartilage-signature genes. (d-g) Datarepresent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001; by ANOVA.

FIGS. 2A to 2G are the results showing that pro-anabolic effect(confirmation of cartilage matrix gene expression) of tankyraseinhibition is mediated by a β-catenin-independent pathway. (a) TOPFlashβ-catenin reporter assay in chondrocytes with activated β-catenin signalpathway after control shRNA or shTnks and shTnks 2 transfection (n=7).(b) Lysates of chondrocytes with activated β-catenin signal pathway wereimmunoblotted for β-catenin after treatment with control or Tnks andTnks2 siRNAs. (c) TOPFlash β-catenin reporter assay in chondrocytes withactivated β-catenin signal pathway following Tnks inhibitory drugtreatment (10 μM, 48 h; n=5). (d) Lysates of chondrocytes with activatedβ-catenin signal pathway were immunoblotted for β-catenin aftertreatment with the indicated Tnks inhibitory drugs for 48 h. β-catenin.(e) mRNA levels of cartilage matrix genes in chondrocytes transfectedwith control, Tnks and Tnks2, or Ctnnb1 siRNAs (n=4). (f) TOPFlashβ-catenin reporter assay in chondrocytes with activated β-catenin signalpathway after 24 h of iCRT 14 treatment as a direct inhibitor ofβ-catenin (n=4). (g) Levels of cartilage matrix proteins in chondrocyteswith activated β-catenin signal pathway treated with iCRT 14 for 24 h asa direct inhibitor of β-catenin. (a-d, f) Wnt-3a recombinant protein wasadded 24 h before harvest. (a, c, e, f) Data represent means±s.e.m.*P<0.05, **P<0.01, ***P<0.001; by ANOVA. * P<0.05, ** P<0.01, ***P<0.001; ANOVA (b, d, g).

FIGS. 3A to 3O are the results showing that Tankyrase interacts withSOX9 and regulates its protein stability and it directly interact withSOX9 and the changes of the concentration of SOX9 when Tankyrase issuppressed. (a) Flowchart of tankyrase substrate identification inchondrocytes. (b) Venn diagram illustrating the overlap oftankyrase-binding proteins identified by three biological replicates(BR) using LC/MS-MS. (c) Histogram of the maximum TTS of the identifiedtankyrase-binding proteins. ▾ indicates the bin that includes proteinsbelonging to the chondrogenesis protein set defined by IPA. (d) Heatmapof TTS and disorder score of TBDs from the predicted tankyrase-bindingproteins having a maximum TTS of ≥0.385 and belonging to the IPAchondrogenesis protein set. The cutoff of 0.385 is the TTS of thetankyrase-binding motifs of mouse AXIN1 and AXIN2. (e)Coimmunoprecipitation of endogenous TNKS with SOX9 in chondrocytes. (f)in situ proximity ligation assay (PLA) to detect interaction betweenendogenous TNKS and endogenous SOX9 in primary cultured mousechondrocytes. Red signals indicate the interactions of endogenousTNKS-SOX9. DAPI was used for counter-staining for nuclei. Scale bar: 25μm (top), 10 μm (bottom). (g) Pull-down assays of GFP-tagged TNKS orTNKS2 with HA-tagged SOX9 in HEK293T cells. (h) Schematic representationof the predicted TBDs in human SOX9 protein (top) and sequence alignmentof TBD1 and TBD2 of SOX9 among vertebrates (bottom). Colored lettersindicate the consensus amino acid sequence of TBDs. (i) Superimpositionof TNKS2:3BP2 and TNKS2:MCL1 complexes with TNKS2 bound to SOX9-TBD1/2.(j) Pull-down assays of Myc-tagged TNKS2 with HA-tagged wild-type SOX9or TBD1 or TBD2 deleted SOX9 mutants in HEK293T cells. (k) Pull-downassay of TNKS2 with wild-type or TBD1/2-deleted SOX9 in HEK293T cells. 1PARylation of wild-type or TBD1/2-deleted SOX9 in HEK293T cells. (l)PARylation assay of wild type or TBD1/2 deleted SOX9 in HEK293T cells.(m) SOX9 immunoblots in chondrocytes after siTnks and siTnks2 treatment.(n) SOX9 immunoblots in chondrocytes after drug treatment. (o)Cycloheximide (CHX) chase analysis of wild-type or TBD1/2-deleted SOX9in HEK293 cells.

FIGS. 4A to 4G are the results showing that RNF146 does not regulateSOX9 activity and cartilage matrix anabolism. (a) TOPFlash β-cateninreporter assay in chondrocytes with β-catenin signal pathway activatedafter control shRNA or shRnf146 transfection=5). (b) Lysate ofChondrocyte with β-catenin signal pathway activated were immunoblottedfor β-catenin after treatment with control siRNA or siRnf146. (a, b)Recombinant Wnt-3a was added 24 h before harvest. (c) 4×48-p89SOX9-dependent Col2a1 luciferase reporter assay in chondrocytestransfected with control shRNA, shRnf146, or shTnks and shTnks2 (n=8).(d) mRNA levels of cartilage-specific matrix genes in mouse chondrocytestreated with control siRNA, siRnf146, or siTnks and siTnks2 (n=5). (e,f) Protein levels of (e) cartilage-specific matrix genes or (0 SOX9 inmouse chondrocytes treated with control siRNA or siRnf146. (g) Factorloadings plot of 14 cartilage matrix genes in the cartilage of 16 BXDmouse strains in terms of transcript abundance, with Rnf146 added to theplot. (a, c, d) Data represent means±s.e.m. * P<0.05, ** P<0.01, ***P<0.001; ANOVA. (b, e, f).

FIGS. 5A to 5J are the results showing that Tankyrase inhibitionenhances cartilage matrix gene expression in a SOX9-dependent manner,indicating that the synthesis of cartilage matrix is occurring throughthe regulation of Sox9 as shown above. (a-c) 4×48-p89 SOX9-dependentCol2a1 luciferase reporter assay in chondrocytes treated with (a)control shRNA, shTnks, shTnks2, or shTnks and shTnks2 (n=8), (b) XAV939,IWR-1, or PARP1/2 inhibitor ABT-888 (n≥5), or (c) 10 μM of varioustankyrase inhibitors for 48 h (n=3). (d) 4×48-p89 SOX9-dependent Col2a1luciferase reporter assay in chondrocytes transfected with control mockvector, wild-type TNKS2 vector, or PARP-dead (PD) TNKS2 mutant vector(n=3). (e) Box plot of fold changes of SOX9 target genes and other genes(two-tailed t test). SOX9 target genes are listed in Table 7. 4×48-p89SOX9-dependent Col2a1 luciferase reporter assay in HEK293T cellstransfected with mock vector or CMV-driven SOX9 expression vector andtreated with (0 siTNKS and siTNKS2 (n=3) or (g) DMSO, XAV939, IWR-1, orABT888 (n=4). (h) 4×48-p89 SOX9-dependent Col2a1 luciferase reporterassay in HEK293T cells expressing wild-type SOX9 or SOX9 withtankyrase-binding motif point mutation (n=6). SOX9 R2A mutant has bothR257A and R271A mutations. (i) Knockdown efficiency of various Sox9siRNAs in primary cultured mouse chondrocytes (n=3). siSox9 #2 was usedthroughout this study. (j) mRNA levels of cartilage-specific matrixgenes in mouse chondrocytes transfected with control siRNA or siSox9 #2followed by DMSO or XAV939 treatment for 72 h (n=6). (a-d, f-j) Datarepresent means±s.e.m. * P<0.05, ** P<0.01, *** P<0.001; ANOVA.

FIGS. 6A to 6G are the results showing Tankyrase inhibition amelioratesOA development in mice. (a, b) GSEA with OA-associated genesets in mousechondrocytes treated with (a) siTnks and siTnks2 versus control siRNA(a) or tankyrase inhibitors versus vehicle (b). (c) Schematicillustration of the DMM model and drug treatment schedule. (d-g)Tankyrase inhibitors protect articular cartilage in surgicallyinduced-OA mouse model. Cartilage destruction assessed by (d) Safranin Ostaining, (e) OARSI grade, and (f) immunostaining of cartilage matrixproteins or (g) SOX9. Scale Bar, (d) 500 μm, (f, g) 25 μm. (e) Datarepresent means±s.e.m. *** P<0.001; Kruskal-Wallis test.

FIGS. 7A to 7H are the results showing that Tankyrase inhibitionstimulates chondrogenic differentiation of mesenchymal stem cells invitro and in vivo. (a) Alcian Blue staining and absorbance quantitationof micromass cultured limb-bud mesenchymal cells treated with theindicated drugs (n=4). Scale bar: 1 mm (top), 300 μm (bottom). (b, c)Histology of hMSC pellets (b) treated with the indicated drugs or (c)infected with the indicated shRNA lentiviruses. Scale bars, 100 μm(top). (d) Knockdown efficiency of shTNKS and shTNKS2 in hMSC (right;n=4). (e-h) Tankyrase knockdown in mesenchymal stem cells regeneratearticular cartilage in vivo. (e) Gross appearance (top) and histologicalimages (middle and bottom) of cartilage lesions. ▾ indicates the graftsites. (f-h) Cartilage regeneration as evaluated using the (f) ICRSmacroscopic score system (n=6), and (g) immunostaining of SOX9 and (h)cartilage matrix proteins in repair tissues in the defects. Scale bar,(g, h) 25 μm, (a, d, f) Data represent means±s.e.m. *P<0.05, **P<0.01,***P<0.001; ANOVA (a, f) t test (d).

FIG. 8 is the result showing that cartilage matrix genes are notinter-correlated in non-cartilaginous organs. Heatmaps of Pearsoncorrelation coefficients of transcript levels for cartilage matrix genesin bone femur, kidney, lung, and brain.

FIGS. 9A to 9D are the results showing that Tankyrase inhibition elicitscartilage-specific transcriptomic profile. (a) Volcano plots of geneexpression changes in mouse chondrocytes treated with siTnks and siTnks2or tankyrase inhibitors. Red dots represent genes with a fold-changeof >3 and a FDR q of <1×10⁻⁵. Blue dots represent genes with afold-change of <1/3 and a FDR q of <1×10⁻⁵. (b) Hierarchical clusteringof fold changes of genes differentially expressed in chondrocytes in atleast one condition (siTnks+siTnks2, XAV939, or IWR-1) compared torespective controls. RNA-Seq was conducted with three biologicalreplicates (BR). (c) GO analysis on differentially expressed genesupregulated in all three conditions (siTnks+siTnks2, XAV939, and IWR-1).(d) Fold change heatmap of cartilage-signature genes in mousechondrocytes treated with siTnks and siTnks2 or tankyrase inhibitors.List of cartilage-signature genes is provided in Table 8.

FIGS. 10A to 10D are the results showing that Tankyrase inhibitioninverts gene expression profiles associated with OA cartilage. (a, b)Fold change heatmaps of OA-associated genes in mouse chondrocytestreated with siTnks and siTnks2 or tankyrase inhibitors. Genes that areupregulated and downregulated in OA cartilage are listed in Tables 9 and10, respectively. (c) Heatmap of Pearson correlation coefficientsbetween transcript levels of Tnks or Tnks2 and catabolic genes in thearticular cartilage of 16 BXD mouse strains. (d) Correlation betweenTnks or Tnks2 and catabolic regulators mRNA levels in the articularcartilage of 16 BXD mouse strains.

FIGS. 11A to 11F are the results showing that Tankyrase inhibitionprevents progression of OA. (a) Light-emitting diode (LED) andfluorescence images of mouse knee joints intra-articularly injected withcarrier-free DiD or DiD-loaded ascorbyl palmitate hydrogel. Images wereacquired on the indicated days after injection. IR shows IR carrier-freeimmediate release and CR shows controlled release after hydrogelinjection. (b) Experiments were done as in (a). Fluorescence images ofmouse femur (femoral condyle) and tibia (tibial plateau) withcarrier-free DiD or DiD-loaded ascorbyl palmitate hydrogel. Images wereacquired at 9 days after IA injection (c) Immunostaining of MMP13 andβ-catenin in articular cartilage of DMM-operated mouse. Scale bars: 50μm. The percentage and the number of immunopositive cells are indicated.(d) Schematic representation of controlled drug delivery to DMM-operatedmice started at 6 weeks after OA operation. (e, f) Cartilage destructionassessed by (e) Safranin O staining (scale bar: 200 μm) and (f) OARSIgrade. Data represent means±s.e.m. *P<0.05; Mann-Whitney U test.

FIGS. 12A and 12B are the results showing that Tankyrase inhibitionstimulates chondrogenic differentiation of mesenchymal stem cells invivo. (a) hMSCs infected with control shRNA lentivirus or TNKS shRNA andTNKS2 shRNA lentiviruses were implanted in the full-thickness cartilagelesions of rat knee joints with fibrin gel constructs. A fibrin-onlygroup was used as a control. Gross appearance of the indicated groups 8weeks after transplantation. Transplantation of hMSCs with TNKS andTNKS2 knockdown resulted in superior healing, filling lesions withcartilage-like tissues. (b) Cartilage repair was assessed using variouscriteria of the ICRS visual histological score system for in vivorepaired cartilage (n=6). Data represent means±s.e.m. *P<0.05; ANOVA.

FIG. 13 is a schematic representation of the molecular mechanismsunderlying the therapeutic effects of Tankyrase inhibitors in OAdiscovered herein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure is based on the discovery that TNKS (Tankyrase)functions as an upstream regulator of SOX9 which is known as animportant player in the formation of cartilage matrix in chondrocytes.Specifically, in the present disclosure, it was identified that TNKSPARsylates SOX9, and the PARsylated SOX9 is then degraded through anintracellular protein degradation mechanism thus lowering theconcentration of SOX9 in the cells. As a result, this makes it difficultfor chondrocytes to synthesize cartilage specific matrix. In addition,it was identified herein the regulatory mechanism downstream of E3Ubiquitin protein ligase involved in the degradation of SOX9.Furthermore, through the suppression of the mechanism using Tankyraseinhibitors that affect the mechanism identified herein, it wasidentified herein the effects of cartilage regeneration, arthritistreatment, and differentiation into chondrocytes.

Thus in one aspect of the present disclosure, there is provided a methodof treating arthritis, or promoting cartilage regeneration or promotingdifferentiation of stem cells into chondrocytes. in a subject in needthereof comprising the step of administering to the subject an effectiveamount of an inhibitor of Tankyrase; or an modified adult stem cell inwhich the expression of Tankyrase is suppressed or a Tankyrase gene isknocked out, wherein the inhibitor of Tankyrase or the modified adultstem cell stabilizes the Sox9 protein or increases the concentration ofthe Sox9 protein by inhibiting the Tankyrase activity promoting thedegradation of Sox9 protein.

Tankyrases (TNKS) is one of the 17 member of ARTD (Diphtheria toxin-likeADP-ribosyltransferase) enzyme superfamily (EC 2.4.2.30), and the ARTDis divided Polymerase (pARTD:ARTD1-6), and monotransferase (mARTD:ARTD7,8, 10-12, 14-17) and inactive enzyme (ARTD9, 13) depending on the kindof amino acid present on the active site.

Human Tankyrase 1 (telomeric repeat binding factor 1 (TRF1)-inter-actingankyrin-related ADP-ribose polymerase; TNKS1/ARTD5/PARP5a) and Tankyrase2 (TNKS2/ARTD6/PARP5b) are multidomain protein having 1327 [NCBI DB:NP_003738.2] and 1166 [NCBI DB: NP_079511, AF329696.1] amino acids,respectively. Particularly, they have a catalytic domain at theirC-terminal called ARTD responsible for ADP-ribosyltransferase activity.Human ARTD is also known as poly(ADP-ribose)polymerases (PARP), TNKS1and TNKS2 have highly conserved sequences and 89% sequence identity. Theconserved SAM domain is located N-terminal of ARTD domain and isinvolved in the formation of homo or hetero oligomers. Tankyrases alsocomprises ankyrin repeat consisting of five ankyrin repeat clusterinvolved in protein-protein interaction.

Particularly ARTD hydrolyze NAD+ (Nicotinamide adenine dinucleotide,oxidized form) into ADP-ribose (ADPr) and nicotinamide. After thehydrolysis, the nicotinamides are released from the binding site of ARTDand involved in the post-translational modification of proteins byattaching several ADP-ribose molecules to target proteins (Lehtio etal., Pharmacology of ADP ribosylation, Vol 280, pp 3576-3593).

In the present disclosure, it was identified that Tankyrase functions asan upstream regulator of SOX9. Tankyrase induces the degradation of SOX9protein through PARsylation (poly(ADP-ribosyl)ation) thereof. In themeantime, SOX9 is known as a master transcription factor important inthe formation of cartilage matrix such as collagen type 2 and Aggrecanin chondrocytes (Ng L J, Wheatley et al. Dev Biol. 1997; 183(1):108-21;Lefebvre V, et al. EMBO J. 1998; 17(19):5718-33; Wright E et al. NatGenet. 1995; 9(1):15-20; Ohba S, et al. Cell Rep. 2015; 12(2):229-43).

Therefore, the control of SOX9 by regulating, particularly suppressingthe upstream regulator Tankyrase can be utilized effectively fortreating various disease or symptoms in which cartilage regenerationprovides an effective treatment.

In one embodiment, the regeneration of cartilage is possible bypromoting stem cells into chondrocytes. In one embodiment, using a mousemodel with inducing degenerative arthritis, it was shown here that theinjection of a tankyrase inhibitor through intraarticular promotes theregeneration of cartilage compared to a control group. Also, it wasconfirmed here that the stem cells with a genetic modification tosuppress Tankyrase expression injected in a rat model with cartilagedefects effectively are differentiated into chondrocytes regeneratingcartilage.

In the present disclosure, the disease which requires a cartilageregeneration for effective treatment is osteoarthritis. Osteoarthritisis also commonly called degenerative arthritis. This is a disease inwhich the joint cartilage surrounding the joint surface of the bone isworn out exposing the bone under the cartilage, and the synovialmembrane around the joint is inflamed, causing pain and deformation, andcartilage regeneration is essential for treatment.

In one embodiment, the Tankyrase inhibitor according to the presentdisclosure inhibits catalytic activity of the ARTD domains of TNKS1 andTNKS2. Therefore, as the Tankyrase inhibitor according to the presentapplication, various inhibitors affecting the ADP-ribosyltransferaseactivity of ARTD or PARP can be used.

In one embodiment, the Tankyrase inhibitor is a substance that binds toa nicotinamide sub-region, adenosine sub-region, or both, which asub-domain of the ARTD domain that is the catalytic region of theTankyrase protein, or substance with tankyrase inhibitory function butthe binding region of which is not identified. The sub-regions are knownbefore (Lehtio et al., Pharmacology of ADP ribosylation, Vol 280, pp3576-3593).

For example, the Tankyrase inhibitors that bind to nicotinamidesub-region are XAV939{3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one}or MN-64 (2-[4-(1-Methylethyl)phenyl]-4H-1-benzopyran-4-one); theadenosine sub-region binding inhibitors are IWR-1[4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide],JW55{N-[4-[[[[Tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl]methyl]amino]carbonyl]phenyl]-2-furancarboxamide},WIKI42-[3-[[4-(4-Methoxyphenyl)-5-(4-pyridinyl)-4H-1,2,4-triazol-3-yl]thio]propyl]-1Hbenz[de]isoquinoline-1,3(2H)-dione,TC-E5001(3-(4-Methoxyphenyl)-5-[[[4-(4-methoxyphenyl)-5-methyl-4H-1,2,4-triazol-3-yl]thio]methyl]-1,2,4-oxadiazole)or G007-LK[(E)-4-(5-(2-(4-(2-chlorophenyl)-5-(5-(methylsulfonyl)pyridin-2-yl)-4H-1,2,4-triazol-3-yl)vinyl)-1,3,4-oxadiazol-2-yl)benzonitrile];and the inhibitors with unidentified binding region are G244-LM{3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazinyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one},or AZ6102{rel-2-[4-[6-[(3R,5S)-3,5-Dimethyl-1-piperazinyl]-4-methyl-3-pyridinyl]phenyl]-3,7-dihydro-7-methyl-4H-pyrrolo[2,3-d]pyrimidin-4-one},or isomers or derivatives thereof are included herein, without beinglimited thereto. The skilled person in the art would be able to selectappropriate inhibitors or isomers or derivatives thereof consideringwhat is disclosed herein.

In other embodiment, Tankyrase inhibitors which may be employed hereinare siRNA (small interfering RNA) or shRNA (small hairpin RNA) or miRNA(microRNA). The siRNA, shRNA and miRNA are silencing mRNA transcriptsthrough RNA interference by forming RISC (RNA Induced Silencing Complex)in which siRNAs sequence specifically bind to the mRNA transcripts.siRNA, shRNA and miRNA have a sequence significantly complementary totheir target sequence. The term significant complementarity means asequence having at least about 70%, about 80%, about 90%, or about 100%complementary to at least 15 consecutive bases of a target sequence.Various antisense oligonucleotides, siRNA, shRNA and/or miRNA targetingTankyrase from various sources may be used for the present disclosure aslong as they bind to a target sequence to silence them. Also biologicalequivalent, derivatives and analogues thereof are also included.Antisense oligonucleotides is a short synthetic nucleotides known in theart, and they bind to a coding sequence of a target protein andsuppress/decrease the expression level of a target protein. AntisenseRNA may have an optimum length according to the methods of transfer ortypes of target genes and be for example 6, 8 or 10 to 40, 60 or 100bases in length. In one embodiment, siRNA is used to suppress theexpression of Tankyrase gene. In one embodiment, sequences of suchsiRNAs are represented by SEQ ID Nos: 25 and 26 for sense and antisense,respectively for TNKS1, and SEQ ID Nos: 27 and 28 for sense andantisense, respectively for TNKS2.

The above sequences may be used as dsRNA in which a sense and antisensesequences bind to each other. Further such sequences may furthercomprise at its 3′ terminal dTdT overhang. As described in FIG. 5, suchsiRNAs effectively suppress the expression of TNKS1/2 at the cellularlevel and thus increasing the concentration of SOX9. This indicates thatsiRNAs can be effectively used for the treatment of cartilageregeneration and arthritis.

As used herein, the terms “treat,” “treatment,” and “treating” includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, and/or reducing severity, progression and/or durationthereof, and/or preventing additional symptoms, and includesprophylactic and/or therapeutic measures. The disease or symptomsincludes disease or symptoms that requires cartilage regeneration foreffective treatment.

The terms “individual,” “subject,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

The present composition may further include one or more pharmaceuticallyacceptable carriers, which includes but does not limited to, saline,sterilized water, Ringer's solution, buffered saline, dextrose solution,maltodextrin solution, glycerol, ethanol, liposome. If desired, thecomposition may further include antioxidant, buffer, antibacterialagents, and other additives known in the art to prepare pharmaceuticalcompositions. The present composition may be formulated into injectableformulations or oral formulations such as capsules, granules, or tabletsby methods known in the art using one or more of diluents, dispersingagents, surfactants, binders and lubricants. Also encompassed for thepresent invention is a target specific composition combined with anantibody or other ligands that specifically recognize a molecule presenton a target tissue or organ of interest. Further latest edition ofRemington's Pharmaceutical Science (Mack Publishing Company, Easton Pa.)may be referred for the preparation and formulation of pharmaceuticalcomposition.

The present composition can be administered by various routes known inthe art such as oral or parenteral delivery for example intravenous,subcutaneous, or intraperitoneal injections or delivery through patch,nasal or respiratory patches. In one embodiment, injections arepreferred. Desirable or optimal dosage may vary among patients dependingon various factors such as body weight, age, sex, general condition ofhealth, diet, severity of diseases, and excretion rate. Dosages used forknown TNKS inhibitors may be referred. Where siRNA, miRNA, antisenseoligonucleotides, shRNA are used, parenteral deliveries are preferred.The typical unit dosage includes but does not limit to for example about0.01 mg to 100 mg a day. Typical daily dosage ranges from about 1 μg to10 g and may be administered one or multiple times a day.

In other aspect, the present disclosure relates to a composition or celltherapy agent for treating arthritis comprising stem cells geneticallymodified to suppress the expression of Tankyrase.

As disclosed in FIGS. 7 and 12, the stem cells modified to suppress theexpression of Tankyrase and the stem cells in which Tankyrases genes areknocked out is able to treat arthritis.

In the present disclosure, the suppression of Tankyrase includes thesuppression of the transcription of Tankyrase genes into mRNAs ortranslation of Tankyrase mRNA into proteins or both.

In one embodiment, the suppression may be accomplished by using shRNAspecific to Tankyrase. In other embodiment, Tankyrase gene may beknocked out. A skilled person in the art would be able to selectappropriate methods to suppress the expression of Tankyrase in stemcells in consideration of the conventional knowledge in the art and whatis disclosed herein.

In the present disclosure, the term “Mesenchymal Stem Cell (MSC)” refersto an adult stem cell which is a pluripotent or multipotent cellobtained from various part of an adult body such as cord blood, bonemarrow, blood, dermis, or periosteum. It can differentiate intocartilage cells. The mesenchymal stem cell may be from an animal,preferably a mammal, more preferably a human mesenchymal stem cell. Moreparticularly, it may be stem cells present in cartilage.

The process of obtaining mesenchymal stem cells is known in the art. Themesenchymal stem cells are isolated from a human or mammalian,preferably human mesenchymal stem cell source. Then the isolated cellsare incubated in an appropriate medium. During the culture, thesuspended cells are removed and the cells attached to the culture plateare passaged to obtain finally established mesenchymal stem cells. Themesenchymal stem cells can be identified, for example, through flowcytometry.

The composition of the present disclosure may be referred to a celltherapeutic agent. The term cell therapeutic agents refer to a medicinewhich is prepared by modifying the cells of autologous, allogenic orxenogeneic origin in vitro using biological, chemical or physicalmethods such as proliferation or selection, in which the cells are usedas a therapeutic agent to replace or repair defect cells in the body.The cell therapeutic agents are controlled as a medicine in US from 1993and in Korea from 2002.

The MSC which may be comprised in the present cell therapeutic agent maybe of autologous, allogenic or xenogeneic origin. More preferably, it isautologous.

In one embodiment, MSC which may be comprised in the present celltherapeutic agent is from animal, preferably mammals, more preferablyfrom human beings.

The route of administration of a cell therapeutic agent or apharmaceutical composition comprising cells according to the presentapplication can be administered through any general route as long as itcan reach the target tissue. Parenteral administration may be, forexample, intraperitoneal administration, intravenous administration,intramuscular administration, subcutaneous administration, intradermaladministration, but is not limited thereto. In one embodiment, thecomposition according to the present invention may be administered in amanner that is intravenously administered or injected directly into anorgan in need of administration of a cell or composition according tothe present invention.

The present composition may be formulated with pharmaceuticallyacceptable carriers. Pharmaceutically acceptable carriers include, forexample, carriers for parenteral administration such as water, suitableoils, saline, aqueous glucose and glycols, and may further includestabilizers and preservatives. Suitable stabilizers include antioxidantssuch as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.Suitable stabilizers include antioxidants such as sodium hydrogensulfite, sodium sulfite or ascorbic acid. Suitable preservatives includebenzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. Inaddition, the composition for cell therapy according to the presentinvention, if necessary, depending on the method of administration orformulation, suspending agent, solubilizing agent, stabilizer, isotonicagent, preservative, anti-adsorption agent, surfactant, diluent,excipient, pH adjuster, painless agent, buffers, antioxidants, and thelike. Pharmaceutically acceptable carriers and formulations suitable forthe present invention, including those exemplified above, are describedin detail in Remington's Pharmaceutical Sciences, latest edition.

The composition for cell therapy of the present invention is formulatedin a unit dose form by formulating using a pharmaceutically acceptablecarrier and/or excipient according to a method that can be easilycarried out by a person skilled in the art to which the presentinvention pertains. The composition may also be administered by anydevice capable of transporting the cell therapy agent to the targetcell. The cell therapy composition of the present invention may includea therapeutically effective amount of a cell therapy agent for thetreatment of a disease.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to the amount of a therapy, which issufficient to treat, attenuate, reduce the severity of arthritis such asosteoarthritis, reduce the duration of arthritis such as osteoarthritis,prevent the advancement of arthritis such as osteoarthritis, causeregression of arthritis such as osteoarthritis, ameliorate one or moresymptoms associated with arthritis such as osteoarthritis, or enhance orimprove the therapeutic effect(s) of another therapy. The exact amountof TNKS inhibitor or cell therapeutic agents may vary depending thedesired effects.

The optimal amount can be readily determined by one of skill in the art,including the type of disease, the severity of the disease, the contentof other ingredients in the composition, the type of formulation, andthe patient's age, weight, general health status, sex and diet, It canbe adjusted according to various factors including the time ofadministration, route of administration and secretion rate of thecomposition, duration of treatment, and drugs used simultaneously.

In one embodiment according to the present application, the cell therapyagent may be administered in the knee joint cavity.

It is important to consider all of the above factors and include anamount that can achieve the maximum effect in a minimal amount withoutside effects. For example, the dosage of the composition of the presentinvention may be 1.0×10⁷ to 1.0×10⁸ cells/kg (body weight), morepreferably 1.0×10⁵ to 1.0×10⁸ cells/kg (body weight) based on the activeingredient. However, the dosage may be variously prescribed by factorssuch as the formulation method, the administration method, the patient'sage, weight, sex, food, administration time, administration route,excretion rate, and response sensitivity, and those skilled in the arttaking these factors into consideration, the dosage can be appropriatelyadjusted. The number of times of administration may be one or two ormore times within the range of clinically acceptable side effects, andthe administration site may be administered at one site or two or moresites.

The present disclosure is further explained in more detail withreference to the following examples. These examples, however, should notbe interpreted as limiting the scope of the present invention in anymanner.

EXAMPLES

Methods

In silico analysis of multi-tissue transcriptomes of the BXD mousepopulation. Cartilage (GN208) (Suwanwela, J. et al. Systems geneticsanalysis of mouse chondrocyte differentiation. J Bone Miner Res 26,747-760, doi:10.1002/jbmr.271 (2011), bone femur (GN411) (Zhu, M. et al.Activation of beta-catenin signaling in articular chondrocytes leads toosteoarthritis-like phenotype in adult beta-catenin conditionalactivation mice. J Bone Miner Res 24, 12-21, doi:10.1359/jbmr.080901(2009)), kidney (GN118), lung (GN160) Alberts, R., Lu, L., Williams, R.W. & Schughart, K. Genome-wide analysis of the mouse lung transcriptomereveals novel molecular gene interaction networks and cell-specificexpression signatures. Respir Res 12, 61, doi:10.1186/1465-9921-12-61(2011), and brain (GN123)(Saba, L. et al. Candidate genes and theirregulatory elements: alcohol preference and tolerance. Mamm Genome 17,669-688, doi:10.1007/s00335-005-0190-0 (2006)) data sets were obtainedfrom GeneNetwork (www.genenetwork.org). illuminaMousev1.db 1.26.0(http://bioconductor.org/packages/illuminaMousev1.db/) and mouse4302. db3.2.3 R. (http://bioconductor.org/packages/illuminaMousev1p1.db/) wereused for probe reannotation. For the cartilage and bone femur data sets,a probe that did not overlap with any known SNPs, perfectly and uniquelymatched the target transcript, and also had the highest expression wasused for each transcript. For other data sets, probes having the highestexpression were used for each transcript. Data sets were clustered usinga hierarchically clustered algorithm (complete connection andcorrelation deviated from the center) at cluster 3.0(http://bonsai.hgc.jp/˜mdehoon/software/cluster/software.htm) andcorrelation heatmap was drawn using Perez-Llamas, C. & Lopez-Bigas, N.Gitools: analysis and visualization of genomic data using interactiveheat-maps (PLoS One 6, e19541, doi:10.1371/journal.pone.0019541 (2011)Gitools 2.3.1. IBM SPSS Statistics 24(http://www.ibm.com/analytics/us/ko/technology/spss/). Major componentsanalysis was used to obtain two factors, and the factor points werecalculated using Regression method.

Primary culture of mouse articular chondrocytes. For the primary cultureof mouse articular chondrocytes, cells were isolated from femoralcondyles and tibial plateaus of 4-5-day-old ICR mice, as describedpreviously⁸³. Chondrocytes were maintained in DMEM supplemented with 10%fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/mlstreptomycin, and cells were treated as indicated in each experiment.Transfection was performed with METAFECTENE PRO (Biontex) according tothe manufacturer's protocol. Small interfering RNAs (siRNAs) used forRNA interference (RNAi) in mouse articular chondrocytes are listed inTable 1. All siRNAs, including negative control siRNA, were purchasedfrom Bioneer. Recombinant mouse Wnt-3a (315-20) was purchased fromPeproTech, and recombinant mouse Dkk-1 (5897-DK) was purchased from R&DSystems.

RT-PCR and qPCR. Total RNAs were extracted using TRI reagent (MolecularResearch Center, Inc.). RNAs were reverse transcribed using EasyScriptReverse Transcriptase (Transgen Biotech). Then, cDNA was amplified byPCR or qPCR with the primers listed in Table 2. qPCR was performed withSYBR TOPreal qPCR 2× preMIX (Enzynomics) to determine transcriptabundance. Transcript quantity was calculated using the ΔΔC_(t) method,and Hprt or HPRT1 levels were used as housekeeping controls. The log 2(fold change) value of the cartilage stromal gene of mouse articularchondrocytes treated with siRNA was clustered using a hierarchicalclustering algorithm (mean association and central correlation distance)in 1.0.4 R package. PCA was performed using the same R package.

Whole-cell lysate preparation. Whole-cell lysates were prepared in RIPAbuffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 8.0, 0.5% sodiumdeoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail(Sigma-Aldrich).

Antibodies. Anti-FLAG tag antibody (3165) was purchased fromSigma-Aldrich. Antibodies against GFP (sc-9996), Sox-9 (sc-20095), Sox-9(sc-166505), Tankyrase-1/2 (sc-8337), Tankyrase-1/2 (sc-365897),Ubiquitin (sc-8017), and Actin (sc-1615), normal Mouse IgG(sc-2025),normal rabbit IgG(sc-2027) were purchased from Santa Cruz Biotechnology.Sox-9 (sc-20095) antibody was used only in FIG. 3e, m and Sox-9(sc-166505) antibody was used only in FIG. 3g . Antibodies againstaggrecan (AB1031), type II collagen (MAB8887), and human mitochondria(MAB1273) were purchased from Millipore, and antibodies against Myc tag(2276) and Sox9 (82630) were purchased from Cell Signaling Technology.Prior to detection of aggrecan, samples were treated with chondroitinaseABC (C3667) from Sigma-Aldrich. Anti-β-Catenin antibody (610154) wasobtained from BD Biosciences. Anti-Poly(ADP-ribose) antibody(AG-20T-0001) was purchased from AdipoGen. All primary antibodies wereused according to the manufacturer's protocol.

Transcript inhibitors of Tankyrase, PARP1/2 and β-catenin response.XAV939 (X3004), IWR-1 (I0161), JW55 (SML0630), and WIKI4 (SML0760) wereobtained from Sigma-Aldrich. G007-LK (B5830) were purchased fromApexbio, G244-LM (1563007-08-8) was from AOBIOUS, MN-64 (HY19351) fromMedChem Express, AZ6102 (S7767) from SelleckChem, and TC-E 5001 (5049)from Tocris. Tankyrase inhibitors were classified into three differentclasses depending on their mode of action (Lehtio, L., Chi, N. W. &Krauss, S. Tankyrases as drug targets. FEBS J 280, 3576-3593,doi:10.1111/febs.12320 (2013)

Haikarainen, T., Krauss, S. & Lehtio, L. Tankyrases: structure, functionand therapeutic implications in cancer. Curr Pharm Des 20, 6472-6488(2014)). ABT-888 (11505) was purchased from Cayman, and iCRT 14 (4299)from Tocris.

RNA sequencing (RNA-seq). Primary cultured mouse articular chondrocyteswere treated with DMSO or 10 μM of XAV939 or IWR-1 for 108 h ortransfected with control siRNA or Tnks and Tnks2 siRNAs. Threebiological replicates were used for each group. One microgram ofhigh-quality RNA samples (RIN>7.0) were used to construct RNA-seqlibraries with the TruSeq Stranded mRNA Library Prep kit (Illumina).Libraries were validated with an Agilent 2100 Bioanalyzer. RNA-seq wasperformed on an Illumina HiSeq 2500 sequencer at Macrogen. The sequencereads were trimmed with Trimmomatic⁸⁶ and mapped against the mousereference genome (mm10) using TopHat. Read counts per gene werecalculated using HTSeq⁸⁸. Differential expression analysis was conductedusing the DESeq2 R package⁸⁹. DEGs were selected using a |fold change|cutoff of >3 and a FDR q cutoff of <1×10⁻⁵. DEGs at least one conditionwere clustered with hierarchical clustering algorithm (ward.D linkagewith euclidean distance) using gplots R package. GO analysis wasconducted using Enrichr⁹⁰. Heatmaps of DEGs that are in thecartilage-signature gene set or the osteoarthritis-signature gene setswere drawn with Gitools.

GSEA analysis. Genes were ranked according to the shrunken log₂ foldchange calculated via DESeq2. GSEA (Subramanian, A. et al. Gene setenrichment analysis: a knowledge-based approach for interpretinggenome-wide expression profiles. Proc Natl Acad Sci USA 102,15545-15550, doi:10.1073/pnas.0506580102 (2005)) was performed inpre-ranked mode, with all default parameters, for thecartilage-signature gene set or the osteoarthritis-signature gene sets.A ten-thousand permutations were used to calculate P values.

Generation of a cartilage-signature gene set. Microarray data for nasalchondrocytes at embryonic day 17.5 and rib chondrocytes at postnatal day1 were obtained from GSE69108 (Ohba, S., He, X., Hojo, H. & McMahon, A.P. Distinct Transcriptional Programs Underlie Sox9 Regulation of theMammalian Chondrocyte. Cell Rep 12, 229-243,doi:10.1016/j.celrep.2015.06.013 (2015)). Microarray data for mouseembryonic fibroblasts (MEFs) were obtained from GSM577694, GSM577695,and GSM577696 of GSE23547(Brellier, F. et al. Tenascin-C triggers fibrinaccumulation by downregulation of tissue plasminogen activator. FEBSLett 585, 913-920, doi:10.1016/j.febslet.2011.02.023 (2011)). The limmaR package (Ritchie, M. E. et al. limma powers differential expressionanalyses for RNA-sequencing and microarray studies. Nucleic Acids Res43, e47, doi:10.1093/nar/gkv007 (2015)) was used to compute differentialexpression between nasal chondrocytes and MEFs or between ribchondrocytes and MEFs. The probe with the highest expression was usedfor each transcript. Genes with a fold-change of >5 and a FDR q of<1×10⁻⁵ in both nasal chondrocytes and rib chondrocytes compared to MEFswere selected as cartilage-signature genes. The cartilage-signaturegenes are listed in Table 8.

Immunoprecipitation. Except for FIG. 3f , the cells were pretreated with10 μM MG-132 (A2585) from ApexBio for 6 hrs. Cell lysates were preparedusing EBC200 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40 and1 mM EDTA) supplemented with the protease inhibitor cocktail. Celllysates were used for pulldown with the indicated antibodies and proteinA/G-Sepharose beads (GE Healthcare). For detection of PARylatedproteins, 5 μM of ADP-HPD (118415) from Calbiochem was added to thelysis buffer. For ubiquitin analysis, 100 mM N-ethylmaleimide (E3876)from Sigma-Aldrich was added to the lysis buffer. The mixtures wereincubated at 4° C. overnight and washed 5 times EBC200. The boundproteins were subjected to SDS-PAGE or LC-MS/MS analysis.

Endogenous tankyrase1/2 pulldown and mass spectrometry. Primary culturedmouse articular chondrocytes were grown for 4 days and treated with 10μM MG132 (Apexbio, A2585). Cells were lysed, and lysates were incubatedwith normal rabbit IgG or anti-tankyrase antibody. The bound proteinswere eluted with 8 M urea in 50 mM NH₄HCO₃ buffer, pH 8.2 for 1 h at 37°C., and in-solution digestion was performed as described previously(Kim, J. S., Monroe, M. E., Camp, D. G., 2nd, Smith, R. D. & Qian, W. J.In-source fragmentation and the sources of partially tryptic peptides inshotgun proteomics. J Proteome Res 12, 910-916, doi:10.1021/pr300955f(2013)). Peptide sequencing was carried out by LC-MS/MS on a ThermoUltimate 3000 RSLCnano high-pressure liquid chromatography systemcoupled to a Thermo Q-Exactive Hybrid Quadrupole-Orbitrap massspectrometer. LC-MS/MS raw data were converted into .mzML files usingProteoWizard MSConvert (Chambers, M. C. et al. A cross-platform toolkitfor mass spectrometry and proteomics. Nat Biotechnol 30, 918-920,doi:10.1038/nbt.2377 (2012)), and the MS-GF+ algorithm (Kim, S. &Pevzner, P. A. MS-GF+ makes progress towards a universal database searchtool for proteomics. Nat Commun 5, 5277, doi:10.1038/ncomms6277 (2014))with a parameter file consisting of no enzyme criteria and staticcysteine modification (+57.022 Da) was used for comparison of all MS/MSspectra against the mouse Uniprot database. The final peptideidentifications had <1% false discovery rate (FDR) q, at the uniquepeptide level. Only fully tryptic and semitryptic peptides wereconsidered. For each biological replicate, proteins that were detectedonly once and proteins that were coimmunoprecipitated with normal rabbitIgG were not considered. For proteins detected in more than onebiological replicate, the peptides and proteins are listed in Table 10.The Venn diagram was drawn with eulerAPE (Micallef, L. & Rodgers, P.eulerAPE: drawing area-proportional 3-Venn diagrams using ellipses. PLoSOne 9, e101717, doi:10.1371/journal.pone.0101717 (2014)).

In silico prediction of tankyrase substrate proteins. The 8×20position-specific scoring matrix (PSSM) generated in Guettler et al(Structural basis and sequence rules for substrate recognition byTankyrase explain the basis for cherubism disease. Cell 147, 1340-1354,doi:10.1016/j.cell.2011.10.046 (2011)) was used to calculate a TTS foreach octapeptide in the proteins identified by LC-MS/MS.

${TTS} = \frac{\sum\limits_{{{pos}.} = 0}^{8}{PSSM}_{{pos}.}}{\max\left( {\sum\limits_{{{pos}.} = 0}^{8}{PSSM}_{{pos}.}} \right)}$

The Python code for calculating the maximum TTS for each tankyrasebinding protein is in the Supplementary Source Code. Only those proteinshaving at least one octapeptide with a TTS of ≥0.385 were considered.This cutoff is the TTS of the tankyrase-binding motifs of mouse AXIN1and AXIN2. AXIN1 and AXIN2, known tankyrase substrates (Huang, S. M. etal. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.Nature 461, 614-620, doi:10.1038/nature08356 (2009)), have the lowestmaximum TTS among the known tankyrase substrates, due to the suboptimalamino acids at the 4^(th) and 5^(th) positions (Guettler, S. et al.Structural basis and sequence rules for substrate recognition byTankyrase explain the basis for cherubism disease. Cell 147, 1340-1354,doi:10.1016/j.cell.2011.10.046 (2011). For further screening, thechondrogenesis category in IPA(https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/)was used. The mouse proteins in the IPA chondrogenesis category arelisted in Table 6. For the candidate proteins, IUPred disorder scoreswere calculated for the octapeptides with a TTS of ≥0.385. The heatmapof TTS and IUPred disorder scores for candidate proteins was drawn withGitools 2.3.1 (Perez-Llamas, C. & Lopez-Bigas, N. Gitools: analysis andvisualisation of genomic data using interactive heat-maps. PLoS One 6,e19541, doi:10.1371/journal.pone.0019541 (2011)).

Cell line culture. HEK293 and HEK293T cells were cultured in DMEMcontaining 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin.Transfection was performed with METAFECTENE PRO (Biontex) or PEItransfection reagent (Sigma-Aldrich) according to the manufacturer'sprotocol. The siRNAs used in HEK293T are listed in Table 1. The siRNAsequences targeting TNKS or TNKS2 were described previously (Huang, S.M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wntsignalling. Nature 461, 614-620, doi:10.1038/nature08356 (2009)).

Plasmids. Human SOX9 cDNA (hMU008919) was purchased from Korea HumanGene Bank and subcloned into a pcDNA3-HA plasmid or a p3×FLAG-CMV10plasmid (see Table 3 for PCR primers used for subcloning). To expresshuman SOX9 under TK promoter, Renilla luciferase gene in a pRL-TKplasmid was replaced by 3×FLAG-SOX9. To generate mutant constructs,PCR-mediated mutagenesis was conducted (see Table 4 list of PCR primersused). The GFP-tagged human TNKS plasmid was a gift from Dr. Chang-WooLee, and the Myc-tagged human TNKS2 plasmid was a gift from Dr. JunjieChen. The FLAG-tagged human TNKS2 plasmid and the FLAG-tagged humanTNKS2 M1054V plasmid were gifts from Dr. Nai-Wen Chi (Sbodio, J. I.,Lodish, H. F. & Chi, N. W. Tankyrase-2 oligomerizes with tankyrase-1 andbinds to both TRF1 (telomere-repeat-binding factor 1) and IRAP(insulin-responsive aminopeptidase). Biochem J 361, 451-459 (2002)). The4×48-p89 SOX9-dependent Col2a1 luciferase reporter construct was a giftfrom Dr. Veronique Lefebvre (Murakami, S., Lefebvre, V. & deCrombrugghe, B. Potent inhibition of the master chondrogenic factor Sox9gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 275,3687-3692 (2000)). Human TNKS2 cDNA was subcloned into a pEGFP-C1plasmid to construct a GFP-tagged human TNKS2 plasmid (see Table 3 forprimers used for subcloning). A control shRNA sequence was inserted intothe pLKO.1 puro and pLKO.1 hygro plasmids. Human TNKS and TNKS2 shRNAsequences were inserted into the pLKO.1 puro and pLKO.1 hygro plasmids,respectively. The shRNA sequences targeting human TNKS or TNKS2 were asdescribed previously (Huang, S. M. et al. Tankyrase inhibitionstabilizes axin and antagonizes Wnt signalling. Nature 461, 614-620,doi:10.1038/nature08356 (2009)). Mouse Tnks and Tnks2 shRNA sequenceswere inserted into the pLKO.1 puro and pLKO.1 hygro plasmids,respectively. Mouse Rnf146 shRNA sequence was inserted into the pLKO.1puro plasmid. The shRNA sequence targeting mouse Tnks was as describedpreviously (Levaot, N. et al. Loss of Tankyrase-mediated destruction of3BP2 is the underlying pathogenic mechanism of cherubism. Cell 147,1324-1339, doi:10.1016/j.cell.2011.10.045 (2011)). The primers used togenerate the above plasmids are listed in Tables 3,4 and 5.

in situ PLA. Primary cultured mouse articular chondrocytes were used forin situ PLA. Duolink® PLA was performed according to the manufacturer'sprotocol (Sigma-Aldrich). Antibodies against Sox-9 (sc-166505) andTankyrase-1/2 (sc-8337) were used to recognize endogenous mouse SOX9 andendogenous mouse tankyrase, respectively.

Sequence alignment of TBD1 and TBD2 of SOX9 among vertebrates. For thesequence alignment of TBD1 and TBD2 of SOX9 among vertebrates,NP_000337.1 (Homo sapiens SOX9), NP_035578.3 (Mus musculus SOX9),NP_989612.1 (Gallus SOX9), NP_001016853.1 (Xenopus tropicalis SOX9), andNP_571718.1 (Danio rerio SOX9) were used.

Structural modeling of protein-peptide interactions. GalaxyPepDock¹⁰⁰was used for modeling of the ARC4 domain of human TNKS2 in complex withthe TBD1 or TBD2 peptide of human SOX9. The structures of ARC4:3BP2 (PDBID: 3TWR) and ARC4:MCL1 (PDB ID: 3TWU) were obtained from Guettler etal. (Guettler, S. et al. Structural basis and sequence rules forsubstrate recognition by Tankyrase explain the basis for cherubismdisease. Cell 147, 1340-1354, doi:10.1016/j.cell.2011.10.046 (2011)).

The ARC4 domain of human TNKS2 (PDB ID: 3TWU_A) and MCL1 peptide (PDBID: 3TWU_B) were used as templates. The MCL1 peptide was substituted bythe TBD1 (255-266 aa) or TBD2 (269-280 aa) peptide of human SOX9 anddocked into a complex. The best predicted model for each of ARC4: SOX9TBD1 and ARC4: SOX9 TBD2 was selected. The model structures weresuperimposed with ARC4:3BP2 and ARC4:MCL1 and visualized using theBIOVIA Discovery Studio Visualizer(http://accelrys.com/products/collaborative-science/biovia-discovery-studio/visualization.html).

Cycloheximide chase analysis. HEK293 cells were treated with 100 μg/mlof cycloheximide form Goldbio (C-930-1) for the indicated number ofhours before lysis. Protein samples were subjected to SDS-PAGE toanalyze protein stability.

Reporter gene assay. A firefly luciferase reporter plasmid withSOX9-dependent Col2a1 enhancer elements (Murakami, S., Lefebvre, V. & deCrombrugghe, B. Potent inhibition of the master chondrogenic factor Sox9gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 275,3687-3692 (2000)) was used to quantify the transcriptional activity ofSOX9. To quantify β-catenin transcriptional activity, the TOPFlashreporter plasmid and recombinant mouse WNT-3a (PeproTech, 315-20) wasused. Primary mouse articular chondrocytes or HEK293T cells weretransfected with both a reporter plasmid and a constitutive Renillaluciferase plasmid. Cells were also treated with siRNAs or drugs asindicated. Renilla and firefly luciferase activity were sequentiallymeasured using a Dual Luciferase Assay Kit (Promega). Renilla luciferasewas used as a control.

List of SOX9 target genes in chondrocytes. Based on Oh et al. (SOX9regulates multiple genes in chondrocytes, including genes encoding ECMproteins, ECM modification enzymes, receptors, and transporters. PLoSOne 9, e107577, doi:10.1371/journal.pone.0107577 (2014)) genes with alog ₂(fold change) of <−2 after Sox9 deletion in mouse rib chondrocytesand associated with SOX9 ChIP-Seq peaks in mouse rib chondrocytes wereselected as SOX9 target genes in chondrocytes. The SOX9 target genes inchondrocytes are listed in Table 7.

Generation of osteoarthritis-signature gene sets. Based on Dunn et al(Gene expression changes in damaged osteoarthritic cartilage identify asignature of non-chondrogenic and mechanical responses. OsteoarthritisCartilage 24, 1431-1440, doi:10.1016/j.joca.2016.03.007 (2016)) geneswith a |fold change| of >2 and a FDR q of <1×10⁻⁵ in damaged sites ofarticular cartilage compared to intact sites within the same patientswith osteoarthritis were selected, and converted to mouse nomenclatureusing the biomaRt R package¹⁰². Genes Tables 9 and 10, respectively.

Preparation of hydrogels and in vivo confirmation of controlled releaseof embedded molecules. 6-O-Palmitoyl-l-ascorbic acid (76183) waspurchased from Sigma-Aldrich. Hydrogels were prepared with6-O-Palmitoyl-l-ascorbic acid as described previously (Zhang, S. et al.An inflammation-targeting hydrogel for local drug delivery ininflammatory bowel disease. Sci Transl Med 7, 300ra128,doi:10.1126/scitranslmed.aaa5657 (2015)). DiD percholate (5702)purchased from Tocris was loaded into the hydrogels and used for imagingof controlled release in mouse knee joints. PBS-suspended hydrogel (10μl, PBS:hydrogel=1:1) containing 50 pmol DiD was administeredintra-articularly, and at 1-9 days post-injection, light-emitting diode(LED) and fluorescence images of knee joints were obtained. LuminoGraphII (Atto) was used to acquire the images.

Experimental OA in mice. Eight-week-old male ICR mice were used forexperimental OA. Experimental OA was induced by DMM (Destabilization ofthe medial meniscus) surgery on the right hindlimb, and sham surgery wasconducted on the left hindlimb as a control (Glasson, S. S., Blanchet,T. J. & Morris, E. A. The surgical destabilization of the medialmeniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.Osteoarthritis Cartilage 15, 1061-1069, doi:10.1016/j.joca.2007.03.006(2007)). 10 μl of PBS-suspended hydrogel (PBS:hydrogel=1:1) containingvehicle or 10 nmol drugs was administered intra-articularly.

Histology and immunohistochemistry. Mouse and rat knee joint samples andhuman cartilage samples were fixed with 4% paraformaldehyde overnight at4° C. All samples were deprotected for 2-4 weeks 0.5M EDTA, pH 7.4 at 4°C. and embedded in paraffin. Mouse and rat paraffin blocks weresectioned at a thickness of 6 μm, and human paraffin blocks weresectioned to a thickness of 5 μm. For Safranin O staining, AlcianBlue/Fast Red staining, or immunostaining, sections were deparaffinizedin xylene and hydrated using a graded ethanol series. All mousehistology images were acquired from medial tibial plateau exceptβ-catenin immunostaining images where medial femoral condyle was usedfor imaging. To assess cartilage destruction in DMM mouse model,Safranin O stained samples were graded based on the OsteoarthritisResearch Society International (OARSI) (Glasson, S. S., Chambers, M. G.,Van Den Berg, W. B. & Little, C. B. The OARSI histopathologyinitiative—recommendations for histological assessments ofosteoarthritis in the mouse. Osteoarthritis Cartilage 18 Suppl 3,S17-23, doi:10.1016/j.joca.2010.05.025 (2010)) by three blindedobservers. On the basis of OARSI grading system, we primarily conductedintegrative evaluation focusing on structural changes and proteoglycanloss in articular cartilage as a measure of cartilage destruction. OARSIgrade 0-2 was classified as early stage and grade over 2 as OA latemiddle stage. Cartilage regeneration in osteochondral defect model wasscored according to the International Cartilage Repair Society (ICRS)scoring system (Haikarainen, T., Krauss, S. & Lehtio, L. Tankyrases:structure, function and therapeutic implications in cancer. Curr PharmDes 20, 6472-6488 (2014) and Mainil-Varlet, P. et al. Histologicalassessment of cartilage repair: a report by the Histology EndpointCommittee of the International Cartilage Repair Society (ICRS). J BoneJoint Surg Am 85-A Suppl 2, 45-57 (2003)) by three blinded observers.

Mouse limb-bud micromass culture. For the micromass culture ofmesenchymal cells, limb-bud cells were isolated from E11.5 ICR mouseembryos. 2.0×10⁷ cells/ml were suspended in DMEM supplemented with 10%FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and 15-μ1drops were spotted on culture dishes. After 24 h, cells were treated asindicated for 3 days and subjected to Alcian Blue staining.

Chondrogenic differentiation of human mesenchymal stem cells. hMSCs werepurchased from Lonza and Thermo Scientific. hMSCs were cultured in α-MEMsupplemented with 20% FBS, 100 units/ml penicillin, 100 μg/mlstreptomycin, and 250 ng/ml amphotericin B. To induce chondrogenesis,2.5×10⁵ hMSCs were centrifuged to form a pellet in α-MEM supplementedwith 20% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250ng/ml amphotericin B. After 3 days, the medium was changed tochondrogenic medium consisting of DMEM/F-12 supplemented with 100units/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B,1.25 mg/ml BSA, 1% Insulin-Transferrin-Selenium, 1 mM Sodium pyruvate,50 μM L-aspartic acid, 50 μM L-proline, 100 nM dexamethasone, and 10ng/ml of TGF-β1 with or without indicated drugs. On day 21 (for drugtreatment) or day 28 (for siRNA treatment), cells were harvested andsubjected to Alcian Blue/Fast Red staining.

Generation of control shRNA-infected or shTNKS and shTNKS2-infectedhuman mesenchymal stem cells. psPAX2 and pMD2.G were transfected toHEK293T. After 3 days, cell supernatants were harvested and filteredthrough a 0.45-μm filter. hMSCs were treated with 8 μg/ml polybrene andinfected with the indicated lentiviruses. Twenty-four hours afterinfection, hMSCs were selected with 5 μg/ml puromycin and 200 μg/mlhygromycin for 4 days.

Rat osteochondral defect model. Twelve-week-old male Sprague Dawley ratswere used as the osteochondral defect model. To expose the articularcartilage in the knee joints, a medial parapatellar incision was madeand the patella was slightly displaced toward the medial condyle. Afull-thickness cartilage defect (3 mm×1 mm×1 mm) was created using a1-mm-diameter spherical drill at the surface of the femoral patellargroove. At the same time, hMSCs were suspended in 10 μl of fibrin glue(TISSEEL) by tapping, and implanted on the defect. To avoid immunerejection, cyclosporine A (C988900) from Toronto Research Chemicals wasinjected intra-peritoneally every day. At 8 weeks, rats were sacrificedfor histological analyses.

Statistics. All experiments were carried out independently at leastthree times. All images are representative of at least three independenttrials. For parametric tests, two-tailed Student's t test or one-wayanalysis of variance (ANOVA) followed by Fisher's least significantdifference post-hoc test were used. For nonparametric tests,Mann-Whitney test or Kruskal-Wallis test followed by Mann-Whitney testwere used. All statistical analysis was performed using IBM SPSSStatistics. A P-value <0.05 was considered statistically significant.

TABLE 1 List of siRNA SEQ Gene Strand siRNA sequences Species ID NOTnks #1 S 5′-CACAGAGUCAC Mouse SEQ ID ACUGACUAdTdT-3′ NO: 1 AS5′-UAGUCAGUGUG SEQ ID ACUCUGUGdTdT-3′ NO: 2 Tnks #2 S 5′-GUCUGUCGUUGMouse SEQ ID AGUACCUUdTdT-3′ NO: 3 AS 5′-AAGGUACACAA SEQ IDCGACAGACdTdT-3′ NO: 4 Tnks #3 S 5′-ACAUAGCAGCG Mouse SEQ IDUUACUGAUdTdT-3′ NO: 5 AS 5′-AUCAGUAACGC SEQ ID UGCUAUGUdTdT-3′ NO: 6Tnks2 #1 S 5′-CAGUGUAGUUU Mouse SEQ ID UGAGUCUAdTdT-3′ NO: 7 AS5′-UAGACUCAAAA SEQ ID CUACACUGdTdT-3′ NO: 8 Tnks2 #2 S 5′-CUGUUCUGACUMouse SEQ ID GGUGACUAdTdT-3′ NO: 9 AS 5′-UAGUCACCAGU SEQ IDCAGAACAGdTdT-3′ NO: 10 Tnks2 #3 S 5′-GUGUCUACUUG Mouse SEQ IDUAUCACAUdTdT-3′ NO: 11 AS 5′-AUGUGAUACAA SEQ ID GUAGACACdTdT-3′ NO: 12Ctnnb1 #1 S 5′-GUUUUAGGCCU Mouse SEQ ID GUUUGUAAdTdT-3′ NO: 13 AS5′-UUACAAACAGG SEQ ID CCUAAAACdTdT-3′ NO: 14 Ctnnb1 #2 S 5′-UCUGAACGUGCMouse SEQ ID AUUGUGAUdTdT-3′ NO: 15 AS 5′-AUCACAAUGCA SEQ IDCGUUCAGAdTdT-3′ NO: 16 Ctnnb1 #3 S 5′-GUAAUCUGGAG Mouse SEQ IDACGUGUAAdTdT-3′ NO: 17 AS 5′-UUACACGUCUC SEQ ID CAGAUUACdTdT-3′ NO: 18Rnf146 #1 S 5′-CAGAUACCUCC Mouse SEQ ID GUUGAAGAdTdT-3′ NO: 19 AS5′-UCUUCAACGGA SEQ ID GGUAUCUGdTdT-3′ NO: 20 Rnf146 #2 S 5′-CUCUAGAGCAUMouse SEQ ID CACAGCUUdTdT-3′ NO: 21 AS 5′-AAGCUGUGAUG SEQ IDCUCUAGACrdTdT-3′ NO: 22 Rnf146 #3 S 5′-GUCGACAAGAG Mouse SEQ IDAUUCCUGAdTdT-3′ NO: 23 AS 5′-UCAGGAAUCUC SEQ ID UUGUCGACdTdT-3′ NO: 24TNKS S 5′-GCAUGGAGCUU Human SEQ ID GUGUUAAUUU-3′ NO: 25 AS5′-AUUAACACAAG SEQ ID CUCCAUGCUU-3′ NO: 26 TNKS2 S 5′-GGAAAGACGUA HumanSEQ ID GUUGAAUAUU-3′ NO: 27 AS 5′-UAUUCAACUAC SEQ ID GUCUUUCCUU-3′NO: 28 Sox9 #1 S 5′-GUAAAGGAAGG Mouse SEQ ID UAACGAUUdTdT-3′ NO: 29 AS5′-AAUCGUUACCU SEQ ID UCCUUUACdTdT-3′ NO: 30 Sox9 #2 S 5′-GAGACAUCGGAMouse SEQ ID CAGACCUUdTdT-3′ NO: 31 AS 5′-AAGGUCUGUCC SEQ IDGAUGUCUCdTdT-3′ NO: 32 Sox9 #3 S 5′-GUUUGUUUCCC Mouse SEQ IDUCUCCAAAdTdT-3′ NO: 33 AS 5′-UUUGGAGAGGG SEQ ID AAACAAACdTdT-3′ NO: 34

TABLE 2 List of PCR Primers Primer SEQ ID Gene Strand sequences SpeciesNO Hprt S 5′-AGTCCCAGCG Mouse SEQ ID TCGTGATTAG-3′ NO: 35 AS5′-GTATCCAACAC SEQ ID TTCGAGAGGTC-3′ NO: 36 Tnks1 S 5′-GAAGGAAGGA MouseSEQ ID GAAGTTGCGG-3′ NO: 37 AS 5-AATGAAAGGAG SEQ ID AACCGTGGAAC-3′NO: 38 Tnks2 S 5′-CGGCGTCTTC Mouse SEQ ID AACAGATACA-3′ NO: 39 AS5′-AGCCATCAAC SEQ ID CATACCTTCAG-3′ NO: 40 Col2a1 S 5′-ACCTTGGACG MouseSEQ ID CCATGAAAGT-3′ NO: 41 AS 5′-CGGGAGGTCT SEQ ID TCTGTGATCG-3′ NO: 42Comp S 5′-GTAAACACCG Mouse SEQ ID CCACTGATGA-3′ NO: 43 AS 5′-TGGGAGAAGCSEQ ID AGAAGACACC-3′ NO: 44 Col9a2 S 5-GATGGGTCCTC Mouse SEQ IDGTGGCTAT-3′ NO: 45 AS 5′-GTTCCCTTTG SEQ ID GGCCTGTTAT-3′ NO: 46 Col6a3 S5′-TTATGGTGCT Mouse SEQ ID GATGTTGACTGG-3′ NO: 47 AS 5′-ATTGCTGTTGSEQ ID GTTTGGTCGTT-3′ NO: 48 Acan S 5′-CCCAAGCACA Mouse SEQ IDGAGGTAAACAG-3′ NO: 49 AS 5′-CTCACATTGC SEQ ID TCCTGGTCTG-3′ NO: 50 Dcn S5′-AGGCTTCCTA Mouse SEQ ID CTCGGCTGTGA-3′ NO: 51 AS 5′-GTTCGGCGGC SEQ IDATTTGACTTT-3′ NO: 52 Col6a1 S 5′-TGAAAATGTG Mouse SEQ ID ASCTCCTGCTGTG-3′ NO: 53 5′-TGTCCCGTTG SEQ ID AGTGTCAGAA-3′ NO: 54 Col9a1 S5′-AGCTGATGGA Mouse SEQ ID TTAACAGGACC-3′ NO: 55 AS 5′-TTCCCAGGGT SEQ IDCTCCAATAGG-3′ NO: 56 Bgn S 5′-GCATTGAGAT Mouse SEQ ID GGGCGGGAA-3′NO: 57 AS 5′-AGTAGGGCAC SEQ ID AGGGTTGTTG-3′ NO: 58 Chad S 5′-ACAACCGCCTMouse SEQ ID GAACCAACT-3′ NO: 59 AS 5-GGGGAGGGATT SEQ ID CTGTGTCTT-3′NO: 60 Matn3 S 5′-CAGTGTGAGG Mouse SEQ ID GGTTTCTG-3′ NO: 61 AS5′-AGCACCATAA SEQ ID GTTCATAGCC-3′ NO: 62 Ctnnb1 S 5′-CCACAGGATT MouseSEQ ID ACAAGAAGCGG-3′ NO: 63 AS 5′-CCATTCCCAC SEQ ID CCTACCAAGT-3′NO: 64 Rnf146 S 5′-AGCACAGAGA Mouse SEQ ID ATGAACCAGCA-3′ NO: 65 AS5′-TGAAGCACCC SEQ ID TTTACACACAGA-3′ NO: 66 Sox9 S 5′-AAGATGACCG MouseSEQ ID ACGAGCAGGA-3′ NO: 67 AS 5′-ATGTGAGTCT SEQ ID GTTCCGTGGC-3′ NO: 68HPRT1 S 5′-CCTGGCGTCG Human SEQ ID TGATTAGTG-3′ NO: 69 AS 5′-CTTGCGACCTSEQ ID TGACCATCTTT-3′ NO: 70 TNKS1 S 5′-TCAGGGAACG Human SEQ IDATTTTGCTGGA-3′ NO: 71 AS 5′-ACTCTGGGTA SEQ ID TGCCTGTTCTC-3′ NO: 72TNKS2 S 5′-GCGATACCCA Human SEQ ID AS AGGCAGACATT-3′ NO: 735′-AACAAGAGGG SEQ ID CAGAGCAGATGG-3′ NO: 74

TABLE 3  List of PCR primers used for subcloning SEQ Primer Enzyme IDGene Strand sequences Sites Species Plasmid NO SOX9 S 5′-CCGAATTCA EcoRIHuman pcDNA3- SEQ TGAATCTCCTGG XbaI HA- ID ACCCCTTC-3′ SOX9 NO: 75 AS5′-CGTCTAGAT SEQ CAAGGTCGAGTG ID AGCTGTGT-3′ NO: 76 SOX9 S 5′-AAGAATTCGEcoRI Human Pcmv10- SEQ AATCTCCTGGAC XbaI 3xFLAG- ID CCCTTCAT-3′ NO: 77AS 5′-CGTCTAGAT SOX9 SEQ CAAGGTCGAGTG ID AGCTGTGT-3′ NO: 78 SOX9 S5′-AAGCTAGCA NileI Human pTK- SEQ ACCATGGACTAC XbaI 3xFLAG- IDAAAGACCA-3′ NO: 79 AS 5-CGTCTAGATC SOX9 SEQ AAGGTCGAGTGA ID GCTGTGT-3′NO: 80 TNKS2 S 5′-AAAAGCTTG HindIII Human pEGFP- SEQ GATCATGTCGGG BamHITNKS2 ID TCGCCGCTG-3′ NO: 81 AS 5′-AAGGATCCT SEQ TATCCATCGACC IDATACCTTCAGG NO: CCTCATAA-3′ 82

TABLE 4 List of PCR primers used for mutagenesis Muta- Primer genesisSpe- SEQ ID Gene Strand sequences Site cies NO SOX9 S 5′-CAGCCCCCTATCΔTBD1 Human SEQ ID GACTTCCGCGA-3′ 772- NO: 83 AS 5′ CCCCTCTCGCT 792SEQ ID TCAGGTCAGCCT-3′ bp NO: 84 SOX9 S 5′-AGCAGCGACGT ΔTBD2 HumanSEQ ID CATCTCCAACAT-3′ 814- NO: 85 AS 5′-GAAGTCGATAG 834 SEQ IDGGGGCTGTCT-3′ bp NO: 86 SOX9 S 5′-AGCAGCGACGT ΔTBD1/2 Human SEQ IDCATCTCCAACAT-3′ 772- NO: 87 AS 5′-CCCCTCTCGCT 834 SEQ ID TCAGGTCAGCCT-3′bp NO: 88 SOX9 S 5′ CCCTTGCCAGA R257A Human SEQ ID GGGGGGCA-3′ NO: 89 AS5′-TGCCCCCTCTCG SEQ ID CTTCAGGTCA-3′ NO: 90 SOX9 S 5′-GACGTGGACATC R271AHuman SEQ ID GGCGAGCTGA-3′ NO: 91 AS 5′-TGCGAAGTCGAT SEQ IDAGGGGGCTGTCT-3′ NO: 92

TABLE 5 List of PCR primers used for shRNA plasmid construction SEQ IDGene Strand Primer sequences Species NO Control s 5′-CCGGAAACAAGATGAAGSEQ ID AGCACCAACTCGAGTTGGT NO: 93 GCTCTTCATCTTGTTTTTT TTG-3′ AS5′-AATTCAAAAAAAACAAG ATGAAGAGCACCAACTCGAG SEQ ID TTGGTGCTCTTCATCTTGTTNO: 94 T-3′ Tnks S 5′-CCGGGCTAGATGTGTTG Mouse SEQ ID GCTGATATCTCGAGATATCNO: 95 AGCCAACACATCTAGCTT TTTG-3′ AS 5′-AATTCAAAAAGCTAGATGTGTTGGCTGATATCTCG SEQ ID AGATATCAGCCAACACATC NO: 96 TAGC-3′ Tnks2 S5′-CCGGCATCGACACAAGC SEQ ID TGATTAAACTCGAGTTTAAT NO: 97CAGCTTGTGTCGATGTTTTT G-3′ AS 5′-AATTCAAAAACATCGAC MouseACAAGCTGATTAAACTCGA SEQ ID GTTTAATCAGCTTGTGTC NO: 98 GATG-3′ Rnf146 S5′-CCGGATTTCTGCCCAC Mouse SEQ ID GTAACATTACTCGAGTAAT NO: 99GTTACGTGGGCAGAAATTT TTTG-3′ AS 5′-AATTCAAAAAATTTCTG CCCACGTAACATTACTCGASEQ ID GTAATGTTACGTGGGCAG NO: 100 AAAT-3′ TNKS S 5′-CCGGGCCCATAATGATHuman SEQ ID GTCATGGAACTCGAGTTCC NO: 101 ATGACATCATTATGGGCTT TTTG-3′ AS5′-AATTCAAAAAGCCCAT SEQ ID AATGATGTCATGGAACTC NO: 102 GAGTTCCATGACATCATTATGGGC-3′ TNKS2 S 5′-CCGGAAGGAAAGACGT Human SEQ ID AGTTGAATACTCGAGTATTNO: 103 CAACTACGTCTTTCCTTTT TTTG-3′ AS 5′-AATTCAAAAAAAGGAA SEQ IDAGACGTAGTTGAATACTCG NO: 104 GATATTCAACTACGTCTTT CCTT-3′

TABLE 6 List of mouse proteins involved in IPA chondrogenesis Proteinsinvolved in chondrogenesis (52 proteins) ALG2 CR3L2 GRN NFKB2 Q9DAB5SOX12 BMAL1 CREB1 GSK3A NKX32 REL SOX4 BMP2 CTNB1 GSK3B PDGFA RELB SOX9BMP4 CYR61 HHAT PER1 RHOA TF65 BMR1B DHH HIF1A PP2BA SHH TNF12 CANB1ENPP1 HMGB2 PP2BB SIR1 VNN1 CANB2 FGF18 IHH PP2BC SMAD3 WNT3A CBP FGFR3NFAC3 PRGC1 SOMA CHP1 GDF5 NFKB1 PTHR SOX11

TABLE 7 List of target gene of SOX9 in chondrocytes SOX9 target genes inchondrocytes (91 genes) Acan Col9a2 Fzd9 Mgp Rab11fip4 Susd5 Aldh1l2Col9a3 Gfpt1 Mia Rhbdd1 Tprgl Alx1 Colgalt2 Gls Mtss1l Rnf144a Trib3Arsi Cox17 Got1 Ncmap Rtkn Trim47 Atf4 Cp Grb2 Ndufa2 Scin Trpv4B230206H07Rik Cpm Hip1r Oat Sdk2 Ucma B4galnt3 D630045J12Rik Hr Papss2Slc1a5 WSCD2 Bcat1 Dnttip1 Kcns1 Pck2 Slc26a2 Wwp2 Bmp6 Enpp2 Lcn2Pcolce2 Slc38a3 Xylt1 Chadl Extl1 Ldlrad3 Pde4dip Slc39a14 Zfp385bChst11 Fam89a Lect1 Phyh Smpd3 Zfp385c Cmklr1 Fbxo7 Leprel1 Plxnb1 SnorcCol11a1 Fgfr3 Lgals3 Ppp1r1b Sobp Col27a1 Fgfrl1 Loxl4 Ppp2ca Sox6Col2a1 Foxd1 Matn3 Prdx5 Spats2l Col9a1 Fry Mgat4a Prelp Stk39

TABLE 8 Cartilage-signature genes Cartilage-signature genes (235 genes)3632451O06Rik Cd14 Dio2 Fzd9 Lect1 Nptx1 Scrg1 Sort1 Zdbf2 4930523C07RikCdkn1a Dnajb9 Gab1 Lipo3 Nr4a2 Scube3 Sox5 Zfp385b A2m Cgref1 Ecm2 Gfpt2Loxl4 Nr4a3 Sdk2 Sox6 Zim1 Acan Chac1 Edil3 Gjc3 Matn1 Nt5e Sec16b Sox9Adamts3 Chad Efcab1 Glis3 Matn3 Omd Sema3e Sparcl1 Adcy2 Chadl Egr1 Gm22Mdfi Panx3 Sema6a Srgap1 Adgrg1 Chrdl1 Egr2 Gm39701 Mertk Papss2 Serinc5Srgn Airn Chst11 Ehd3 Gm7265 Mfi2 Pcsk6 Sim2 Srxn1 Ak4 Clec3a Eng Gprc5aMfsd7c Pde3a Slc16a2 Stk26 Alx1 Cmklr1 Enpp1 Gpx3 Mgat4a Perp Slc16a4Stk32b Angptl1 Cmtm5 Enpp2 Grb10 Mia Phxr4 Slc1a1 Stk40 Arc Col10a1Epas1 Gstk1 Mir377 Pla2g5 Slc1a5 Sulf2 Arl5b Col11a1 Epyc Hapln1 Mir411Plcd1 Slc22a23 Tcn2 Asb4 Col11a2 Ern1 Hist1h1c Mir505 Plet1 Slc22a4 Tet1Atf3 Col2a1 Extl1 Hivep2 Mir568 Plod2 Slc25a36 Tet2 Atp1b2 Col9a1 F13a1Hpgd Moxd1 Prg4 Slc26a2 Tgfb2 Auts2 Col9a2 Fabp7 Igsf9b Mpzl2 Prkg2Slc2a10 Tmbim1 B4galnt3 Col9a3 Fam180a Il16 Mt2 Prss35 Slc38a3 Tmem56Baiap2l1 Colgalt2 Fam19a5 Islr Mtap7d3 Ptger1 Slc6a12 Tnfrsf21 BC026585Comp Fam46a Itga10 Mtss1l Rab11fip4 Slc7a11 Tns2 Bdh1 Cpm Fbln7 Kank1Mustn1 Rbp4 Slc7a3 Tram2 Bmp2 Cpxm2 Fgfr2 Kcna6 Ncmap Rcan1 Slc8a3Trp53inp2 Bmp5 Creb3l2 Fgfr3 Kcnk1 Ndrg2 Rgs2 Smox Trps1 Bmp6 Crispld1Fmod Kcnma1 Nebl Rin2 Smpdl3a Trpv4 Btg2 Cspg4 Fos Kdm6b Nfatc1 Rnf144bSnora23 Ucma C1qtnf3 Cthrc1 Fosb Kdm7a Nfatc2 S100a1 Snora28 Wisp3 C4bCtsh Frmd4b Kif21a Ngf S100b Snorc Xist Car6 Cybrd1 Fry Klhl13 Ninj1Scara3 Snord82 Xylt1 Cd109 Cytl1 Frzb Klk10 Ninj2 Scin Sobp Zbtb20

TABLE 9 Unregulated genes in osteoarthritic cartilage Upregulated genesin osteoarthritic cartilage (150 genes) 3830406C13Rik Cenpk Fam167aKcnn4 Pcdh10 St6galnac5 Abracl Cep55 Fam60a Kcns3 Pcdh18 Stx1a Adamts14Chst13 Fat3 Kif20a Pgm2l1 Syt11 Adamts5 Cited4 Fgf9 Lamb3 Plaur Sytl2Adamts6 Ckb Fhl2 Lif Plekhg1 Tbx3 Adgrg1 Clic3 Foxf1 Lmo2 Popdc3 Tenm3Adtrp Col13a1 Fstl3 Lrrc8c Postn Tfpi AI661453 Col18a1 Fzd10 Lrrc8ePrex2 Tgfbi Akr1c20 Col1a1 Galnt7 Lum Ptges Tmem100 Anln Col7a1 Gja1Map1b R3hdml Tmem119 Arhgap44 Cpeb2 Gjb2 Mob3b Rab23 Tmem200a Arl4aCsdc2 Glis3 Moxd1 Rcan1 Tmem59l Arntl2 D330045A20Rik Glrb Msx2 Rhbdl2Tnfaip6 Aspm Diras1 Gmnn Mtss1 S100a4 Tnfrsf12a Aspn Dkk3 Gpc4 NcapgSema3c Tom1l1 Atrnl1 Dnajc12 Gria2 Nedd4l Serpine1 Top2a B3gnt2 DnerHey2 Nedd9 Serpine2 Trim36 B3gnt5 Dsg2 Hhipl1 Ngf Sgk1 Uroc1 Bmpr1bDusp4 Hmga2 Nt5e Sik1 Vcan C1galt1 Ebf3 Homer2 Ntf3 Slc2a5 Veph1 Car12Egr2 Hunk Ociad2 Slc38a5 Vwc2 Cdk1 Epha3 Ier3 Ogn Slc6a6 Wisp1 Cdkn2bEva1a Iqgap3 Osbpl3 Slitrk6 Wnt5a Cdkn3 Evi2a Itga3 P3h2 Sntb1 Zfp365Cenpf Fam132b Kcne4 Pamr1 Sqrdl Zfp367

TABLE 10 Downregulated genes in osteoarthritic cartilage Downregulatedgenes in osteoarthritic cartilage (71 genes) Agtr2 Cmya5 Fbln7 Lgi4Ptger3 Srl A1x4 Col11a2 Fgf14 Lrrtm2 Rarres2 Steap4 Apol9b Col16a1 FrzbMpped2 Rcan2 Stk32b Atp1b2 Crim1 Gpc5 Myh14 Rflna Tac1 C530008M17RikCyp39a1 Gprc5b Myoz3 Rspo3 Tceal5 Cacna1c Dact1 Grin2c Nfam1 Sdc3Tmem176a Cacna2d2 Dcc Gucy1a3 Nrxn2 Sez6l Tmem176b Capn6 Ddit4 Hmgcll1Obscn Sgsm1 Tnfrsf4 Cdhr1 Erich3 Igf2 Pde3b Slc14al Wnk2 Ces1a Esr1Il17rb Piezo2 Slc25a27 Zcchc5 Chrdl2 Evx1 Il18bp Ppp1r1b Slitrk4 Zfp385cCmtm5 Fam198a Kif1a Prx Sncg

Example 1. Identification of a Regulatory Factor that Governs CartilageMatrix Anabolism

To screen for a key regulatory factor that could be targeted tostimulate cartilage matrix anabolism, genetic analysis on transcriptomesof mouse reference populations using post-hoc factor analysis wereconducted. First, we assessed the transcriptional variance in thecartilage tissues of 16 strains of BXD mice. We noted that, among 21cartilage matrix genes listed up by Heinegard and Saxne (The role of thecartilage matrix in osteoarthritis. Nat Rev Rheumatol 7, 50-56,doi:10.1038/nrrheum.2010.198 (2011)), 14 cartilage matrix genes showedstrong positive correlation in their transcript abundance (FIG. 1a ).These high correlations were absent in organs without cartilaginousfunctions, such as bone femur, kidney, lung, and brain (FIG. 8). We thenattempted to extract a common axis underlying cartilage anabolism byperforming a principal component analysis on 14 highly inter-correlatedcartilage matrix genes (see black box in FIG. 1a ). The first axisidentified (Factor 1) essentially reflects the state of cartilage matrixanabolism (FIG. 1b ). We then computed Pearson's correlationcoefficients between these 14 cartilage matrix genes and Factor 1 genes(Factor I and transcription factors, enzymes and various gene identifiedas signal molecules with unknown functions in cartilage). Tankyraseshowed striking negative correlations with the anabolic axis and withindividual cartilage matrix genes and was therefore, investigatedfurther (FIG. 1b, c ). Tankyrase showed striking negative correlationswith the anabolic axis and with individual cartilage matrix genes andwas therefore selected as a candidate and investigated further (FIG. 1b,c ).

We then examined the potential regulatory role of tankyrase in cartilageanabolism. Knockdown of both Tnks and Tnks2 collectively induced theexpression of cartilage-specific matrix genes in primary cultured mousechondrocytes (FIG. 1d, e ). On the other hand, the individual knockdownof Tnks or Tnks2 failed to increase the cartilage matrix anabolism,suggestive of the redundant roles of tankyrase-1 and -2 in thisregulation (FIG. 1e ). Treatment with XAV939 or IWR-1, highly specificand potent TNKS/2 inhibitors also increased the expression ofcartilage-specific matrix genes in chondrocytes (FIG. 1f, g ). However,the PARP1/2 inhibitor, ABT-888, failed to increase their expressions.PARP is a member of the family with PARylation activity. PARP 1 to PARP16 are known and TNK1 and TNK2 is PARP-5a, and PARP-5b, respectively.Thus the above result indicates clearly that only TNKS inhibition amongPARP can induce the cartilage matrix specific gene expression sinceXAV939 is a TNKS inhibitor and ABT-888 is a PARP1/2 inhibitor. ABT-888used as a negative control.

To comprehensively elucidate the effect of tankyrase inhibition at thewhole transcriptome level, we performed RNA sequencing for chondrocytestreated with siRNAs targeting Tnks and Tnks2, XAV939, or IWR-1(FIG. 9a). As a result, all three tankyrase inhibition group compared to theirrespective control groups showed similar group of differentiallyexpressed genes up or downregulated (FIG. 9b ). GO analysis of thecommonly upregulated genes in response to tankyrase inhibition revealeda strong association with terms related to cartilage development (FIG.9c ). Next, we generated a comprehensive list of cartilage-signaturegenes by utilizing public transcriptome datasets. Tankyrase inhibitioninduced strong transcription of key cartilage-identity genes (FIG. 9d ).In addition, gene set enrichment analysis (GSEA) revealed thatcartilage-signature genes were positively enriched in the wholetranscriptome obtained from chondrocytes treated with siTnks and siTnks2or tankyrase inhibitors, XAV939 and IWR-1(FIG. 1h, j ). Thus, tankyraseinhibition promotes cartilage matrix anabolism and strengthens overallchondrogenic features in chondrocytes

Example 2. Identification that SOX9 Interacts with Tankyrase Through itsConserved Tankyrase-Binding Domains

Here it was discovered that SOX9 interacts with tankyrase through itsconserved tankyrase-binding domains. To understand the molecularmechanism underlying the effect of tankyrase inhibition on cartilageanabolism, we aimed to identify tankyrase substrates responsible for theregulation of cartilage matrix genes. Axin, a well-established target oftankyrase, is subjected to proteasomal degradation uponPARylation-dependent ubiquitination (Huang, S. M. et al. Tankyraseinhibition stabilizes axin and antagonizes Wnt signalling. Nature 461,614-620, doi:10.1038/nature08356 (2009), Zhang, Y. et al. RNF146 is apoly(ADP-ribose)-directed E3 ligase that regulates axin degradation andWnt signalling. Nat Cell Biol 13, 623-629, doi:10.1038/ncb2222 (2011)).Consistently, tankyrase inhibition reduced β-catenin stability andactivity in chondrocytes (FIG. 2a-d ). However, when transcriptioninhibitor iCRT 1429 responsive to β-catenin or Ctnnb1 siRNA, it wasfound that they did not significantly affect the expression of cartilagematrisome. This indicates that β-catenin is not involved in thetankyrase inhibition in cartilage anabolism (FIG. 2 e, f, g).

To find a novel tankyrase-binding substrate that regulates cartilagematrix anabolism, we performed liquid chromatography-tandem massspectrometry (LC-MS/MS) analysis for the proteome co-immunoprecipitatedwith the endogenous tankyrase in chondrocytes (FIG. 3a and Table 10). Weconsidered proteins that are detected in more than one biologicalreplicate as putative tankyrase-interacting proteins (FIG. 3b ). Amongthese binding partners, candidate substrates were further screened usinga tankyrase-targeting score (TTS) system (Guettler, S. et al. Structuralbasis and sequence rules for substrate recognition by Tankyrase explainthe basis for cherubism disease. Cell 147, 1340-1354,doi:10.1016/j.cell.2011.10.046 (2011)). Ingenuity pathway analysis (IPA)revealed four candidate proteins above the TTS cutoff 0.385) that fellinto the chondrogenesis category (FIG. 3c ). Then IUPred disorder score(Dosztanyi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web serverfor the prediction of intrinsically unstructured regions of proteinsbased on estimated energy content. Bioinformatics 21, 3433-3434,doi:10.1093/bioinformatics/bti541 (2005)) was used to filter unlikelytargets, wherein tankyrase-binding motifs are positioned in a highlystructured region (FIG. 3d ). SOX9 exhibited both high TTS and disorderscores, and selected as a candidate. Endogenous interactions betweentankyrase and SOX9 in chondrocytes were confirmed byco-immunoprecipitation assay and in situ proximity ligation assay (PLA)(FIG. 3e, f ).

Moreover, our cell-based assay indicated that SOX9 binds to bothtankyrase-1 and tankyrase-2 (FIG. 3g ). The two tankyrase-bindingdomains (TBDs) of SOX9, designated as TBD1 and TBD2, are highlyconserved among vertebrates (FIG. 3h ). Based on structural simulations,TBD1 and TBD2 peptides fit into the binding pocket located central tothe ankyrin repeat cluster (ARC) IV domain of tankyrase where knownsubstrates, SH3 domain-binding protein (3BP2) and myeloid cell leukemiasequence 1 protein (MCL1), are aligned (FIG. 3i ). The deletion ofeither TBD1 or TBD2 resulted in the reduction in the binding affinity ofSOX9 for tankyrase (FIG. 3j ), while simultaneous deletion of both TBDsnearly abolished this association (FIG. 3k ).

Example 3. Tankyrase Inhibition Enhances SOX9 Stability and Activity byUncoupling SOX9 from PARylation-Dependent Degradation

Here, we investigated whether tankyrase binding to SOX9 is coupled toPARylation of SOX9. Wild-type SOX9 underwent extensive PARylation,whereas SOX9 mutant missing both TBDs exhibited a markedly reducedPARylation level (FIG. 3l ). Tankyrase-dependent PARylation is generallylinked to the degradation of substrate proteins (Riffell, J. L., Lord,C. J. & Ashworth, A. Tankyrase-targeted therapeutics: expandingopportunities in the PARP family. Nat Rev Drug Discov 11, 923-936,doi:10.1038/nrd3868 (2012)). In fact, tankyrase inhibition promoted SOX9protein expression in chondrocytes (FIG. 3m, n ). and SOX9 TBD mutantshowed augmented stability compared with wild-type SOX9 (FIG. 3o ).Taken together, the disruption of the physical interactions betweentankyrase and SOX9 and the consequent abolishment of SOX9 PARylationresults in the stabilization of SOX9.

To date, RNF146 is the only known E3 ubiquitin ligase that mediatesPARylation-dependent ubiquitination and degradation of substrates(Zhang, Y. et al. RNF146 is a poly(ADP-ribose)-directed E3 ligase thatregulates axin degradation and Wnt signalling. Nat Cell Biol 13,623-629, doi:10.1038/ncb2222 (2011), DaRosa, P. A. et al. Allostericactivation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ationsignal. Nature 517, 223-226, doi:10.1038/nature13826 (2015), Andrabi, S.A. et al. Iduna protects the brain from glutamate excitotoxicity andstroke by interfering with poly(ADP-ribose) polymer-induced cell death.Nat Med 17, 692-699, doi:10.1038/nm.2387 (2011)). In particular, RNF146is best known to regulate tankyrase-dependent Axin degradation andhence, β-catenin stabilization³³. Consistent with this notion, shRNA orsiRNA-mediated knockdown of Rnf146 effectively reduced TOPFlash activityand β-catenin level (FIG. 4a, b ). However, unlike Tnks and Tnks2 doubleknockdown, Rnf146 knockdown in chondrocytes failed to increase SOX9transcriptional activity, the expression of cartilage matrix genes, orSOX9 protein level (FIG. 4c-f ). Our experimental findings were furthersupported by factor analysis results based on mouse referencepopulations. A total of 14 inter-correlated cartilage matrix genesexhibited insignificant correlation with Rnf146 (r=−0.12; P=0.66; FIG.4g ). Our data suggest an intriguing possibility that PAR-dependent E3ligases other than RNF146 may exist and regulate PARylation-dependentSOX9 regulation.

Example 4. Identification that SOX9 is Necessary for TankyraseInhibition-Induced Cartilage Matrix Gene Expression

Here, we used a 4×48-p89 SOX9-dependent Col2a1 enhancer reporter(Murakami, S., Lefebvre, V. & de Crombrugghe, B. Potent inhibition ofthe master chondrogenic factor Sox9 gene by interleukin-1 and tumornecrosis factor-alpha. J Biol Chem 275, 3687-3692 (2000)) to investigatewhether the increase in SOX9 levels with tankyrase inhibition enhancesthe overall transcriptional activity of SOX9. Double knockdown of Tnksand Tnks2 and nine different tankyrase-specific inhibitors specificallyincreased the transcriptional activity of SOX9 in chondrocytes (FIG.5a-c ). Moreover, the overexpression of wild-type TNKS2 resulted in amarked reduction in the transcriptional activity of SOX9, while thecatalytically inactive form of TNKS2 (TNKS2 M1054V) suppressed SOX9activity to a moderate extent (FIG. 5d ).

SOX9 target genes (Oh, C. D. et al. SOX9 regulates multiple genes inchondrocytes, including genes encoding ECM proteins, ECM modificationenzymes, receptors, and transporters. PLoS One 9, e107577,doi:10.1371/journal.pone.0107577 (2014)) were overall upregulated upontankyrase knockdown or inhibition at the whole transcriptome level (FIG.5e ).

Meanwhile, SOX9 is known to bind to its own enhancer and auto-regulateits expression (Mead, T. J. et al. A far-upstream (−70 kb) enhancermediates Sox9 auto-regulation in somatic tissues during development andadult regeneration. Nucleic Acids Res 41, 4459-4469,doi:10.1093/nar/gkt140 (2013)). As disclosed hereinbefore, we thoughtthat Tankyrase are involved in the degradation of SOX0, we furtherinvestigated whether tankyrase regulates SOX9 activitypost-transcriptionally at the protein level. For this, the effect oftankyrase inhibition with abundant amount of SOX9 protein expressed asFIGS. 5f and 5g was analyzed and the luciferase reporter assays usingthe SOX9-dependent Col2a1 enhancer construct in cells constitutivelyexpressing SOX9 mRNA under the control of a cytomegalovirus (CMV)promoter were performed. Tankyrase inhibition using siRNAs or drugsincreased the transcriptional activity of exogenously expressed SOX9 inHEK293T cells (FIG. 5f, g ). Furthermore, point mutations of Arg in thefirst position to Ala in both TBD1 and TBD2 of SOX9 synergisticallyenhanced the transcriptional activity of SOX9 (FIG. 5h ), suggestingthat disruption of the interaction between tankyrase and SOX9 issufficient to enhance the transcriptional activity of SOX9. Cartilagematrix gene expression induced by tankyrase inhibition was completelyabolished by SOX9 knockdown (FIG. 5i, j ). Taken together, SOX9 servesas an essential target of tankyrase for the role of tankyrase as ananabolic regulator in chondrocytes.

Example 5. Tankyrase Inhibition Protects Against OsteoarthriticCartilage Destruction in Mice

Our results disclosed herein suggest that tankyrase may perform aphysiological role in the regulation of cartilage matrix homeostasis. Ascartilage homeostasis is disrupted during OA development. Thus, weinvestigated how tankyrase inhibition affects the expression ofOA-associated genes when cartilage matrix homeostasis is destructedduring OA development. By utilizing public transcriptome datasets, wegenerated a comprehensive list of OA-associated genes that areupregulated and downregulated in OA patients. Notably, OA-associatedgenes upregulated in patients were overall repressed in chondrocytesupon tankyrase inhibition (FIG. 10a ). In contrast, OA-associated genessuppressed in patients were strongly transactivated by tankyraseinhibition (FIG. 10b ). This inverted pattern of gene expressionprofiling was evident even at the whole transcriptome level (FIG. 6a, b).

Next, we assessed the in vivo effects of tankyrase inhibition oncartilage matrix homeostasis in surgically induced OA mouse model. Forthe stable and prolonged delivery of tankyrase inhibitors to mouse kneejoints, we used injectable hydrogels made of ascorbyl palmitate.Intra-articular (IA) injection of this hydrogel-based drug deliverysystem allowed controlled local release of the loaded small molecule toarticular cartilage over 9 days (FIG. 11a, b ). IA administration ofhydrogel-mediated XAV939 or IWR-1, the two representative tankyraseinhibitors with different modes of actions, resulted in a significantreduction in the degeneration of cartilage matrix caused by thedestabilization of the medial meniscus (DMM) (FIG. 6 c, d, e). Aconcomitant increase in type II collagen and aggrecan was observed (FIG.60 and the expression of SOX9 was retained in the cartilage treated withtankyrase inhibitors (Fig. g). In addition, we observed that IA deliveryof tankyrase inhibitors effectively reduced the production of matrixmetalloproteinase 13 (MMP13)(Billinghurst, R. C. et al. Enhancedcleavage of type II collagen by collagenases in osteoarthritic articularcartilage. J Clin Invest 99, 1534-1545, doi:10.1172/JCI119316 (1997)that is a key enzyme involved in the catabolism of Type II collagen.These experimental results are in line with the correlation analysisbased on mouse reference populations, indicating that tankyrase exhibitsa negative and positive correlation with cartilage matrix genes (FIG.1b, c ) and catabolic regulators (FIG. 10d, e ), respectively.

Based on the pro-anabolic effect of tankyrase inhibitors, we tested thepotential of XAV939 to treat late-stage OA cartilage. In the mouse DMMmodel (Kim, J. H. et al. Matrix cross-linking-mediatedmechanotransduction promotes posttraumatic osteoarthritis. Proc NatlAcad Sci USA 112, 9424-9429, doi:10.1073/pnas.1505700112 (2015), earlyosteoarthritic lesions were observed 2 weeks after surgery, while 70% ofmice had reached late-stage OA after 6 weeks from DMM surgery. XAV939administration for additional 6 weeks resulted in the reduction in thecartilage destruction as compared with the vehicle-treated mice, whichexperienced further OA progression (FIG. 11 c, d, e). Taken together,our results indicate that tankyrase inhibitors effectively amelioratecartilage destruction in mice through the attenuation of the imbalancebetween matrix anabolism and catabolism.

Example 6. Tankyrase Inhibition Stimulates Chondrogenic Differentiationof MSCs and Produce Therapeutic Effects

As mesenchymal progenitor cells are responsible for the regenerativecapacity of damaged cartilage (Johnson, K. et al. A stem cell-basedapproach to cartilage repair. Science 336, 717-721,doi:10.1126/science.1215157 (2012), Jiang, Y. & Tuan, R. S. Origin andfunction of cartilage stem/progenitor cells in osteoarthritis. Nat RevRheumatol 11, 206-212, doi:10.1038/nrrheum.2014.200 (2015)), weinvestigated the role of tankyrase in the chondrogenic differentiationof MSCs. The tankyrase inhibitors, XAV939 and IWR-1, effectively inducedchondrogenic nodule formation in micromass cultures of mouse limb-budmesenchymal cells (FIG. 7a ), and both pharmacological inhibition anddouble knockdown of TNKS and TNKS2 effectively enhanced the chondrogenicdifferentiation of hMSCs (FIG. 7 b, c, d).

We next evaluated the effect of tankyrase inhibition on stem cell-basedrestoration of hyaline cartilage. A full-thickness osteochondral lesionwas filled with a fibrin gel containing hMSCs transduced with control orTNKS and TNKS2 shRNAs. After 8 weeks, Defects transplanted withhMSCs-control shRNA failed to fully recover the organization of hyalinecartilage and exhibited features of fibrocartilage (FIG. 7e, f and FIG.12). However, lesions implanted with hMSCs-shTNKS/2 showed regeneratedhyaline cartilage, similar to the articular cartilage with robustexpression of SOX9 and cartilage-specific matrix proteins (FIG. 7g, h ).

Innate MSCs are present in cartilage tissues and there are many MSCs inthe bone marrow and synovial fluid around the cartilage, which may beinvolved in cartilage regeneration. Here it was shown that theinhibition of Tankyrase can lead to the differentiation of MSCs intochondrocytes in cell and mouse cartilage regeneration model. Thisindicates that the promotion of differentiation of MSC into chondrocytesby inhibition of Tankyrase can be advantageously used for cartilageregeneration in degenerative arthritis.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, devices, and materials are described herein.

1. A method of treating arthritis in a subject in need thereofcomprising administering to the subject an effective amount of aninhibitor of Tankyrase; or an modified adult stem cell in which theexpression of Tankyrase is suppressed or Tankyrase gene is knocked out,wherein the inhibitor of Tankyrase or the modified adult stem cellstabilizes the Sox9 protein or increases the concentration of the Sox9protein by inhibiting the Tankyrase activity promoting the degradationof Sox9 protein.
 2. The method of claim 1, wherein the inhibitor ofTankyrase leads to a chondrogenic differentiation of an adult stem cellsleading to chondrogenic regeneration.
 3. The method of claim 1, whereinthe inhibitor of Tankyrase is an agent that binds to a nicotinamidesub-domain of ARTD domain which is a catalytic domain of a Tankyraseprotein, an agent that binds to an adenosine sub-domain of a Tankyraseprotein or an agent that binds to an unidentified domain of a Tankyraseprotein.
 4. The method of claim 3, wherein the agent that binds to anicotinamide sub-domain of ARTD domain is XAV939{3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}or MN-64 {2-[4-(1-methylethyl)phenyl]-4H-1-benzopyran-4-one}; the agentthat binds to an adenosine sub-domain of a Tankyrase protein is IWR-1[4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolynyl-benzamide], JW55{N-[4-[[[[tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl]methyl]amino]carbonyl]phenyl]-2-purancarboxamide},WIKI42-[3-[[4-(4-methoxyphenyl)-5-(4-pyridynyl)-4H-1,2,4-triazol-3-yl]thio]propyl]-1Hbenz[de]isoquinoline-1,3(2H)-dion,TC-E5001{3-(4-methoxyphenyl)-5-[[[4-(4-methoxyphenyl)-5-methyl-4H-1,2,4-triazol-3-yl]thio]methyl]-1,2,4-oxadiazolor G007-LK{(E)-4-(5-(2-(4-(2-chlorophenyl)-5-(5-(methylsulfonyl)pyridine-2-yl)-4H-1,2,4-triazol-3-yl)vinyl)-1,3,4-oxadiazole-2-yl)benzonitrile};and the agent that binds to an unidentified domain of a Tankyraseprotein is G244-LM {3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazynyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}, or AZ6102{rel-2-[4-[6-[(3R,5S)-3,5-dimethyl-1-piperazynyl]-4-methyl-3-pyridynyl]phenyl]-3,7-dihydro-7-methyl-4H-pyrrolo[2,3-d]pyrimidine-4-one},or isomers or derivative thereof.
 5. The method of claim 1, wherein theinhibitor is a siRNA that suppresses the expression of Tankyrase geneinto a Tankyrase protein.
 6. The method of claim 5, wherein the siRNA isa dsRNA consisting of RNAs of SEQ ID NO: X1 and SEQ ID NO:X2; or a dsRNAconsisting of RNAs of SEQ ID NO: X3 and SEQ ID NO:X4.
 7. The method ofclaim 1, wherein the adult stem cell is autologous or allogenic.
 8. Themethod of claim 1, wherein the adult stem cell is a mesenchymal stemcell.
 9. A method of promoting the differentiation of an adult stem cellinto a cartilage cell by treating the stem cell with an inhibitor ofTankyrase.
 10. The method of claim 9, wherein the adult stem cell is amesenchymal stem cell.
 11. The method of claim 2, wherein the inhibitorof Tankyrase is an agent that binds to a nicotinamide sub-domain of ARTDdomain which is a catalytic domain of a Tankyrase protein, an agent thatbinds to an adenosine sub-domain of a Tankyrase protein or an agent thatbinds to an unidentified domain of a Tankyrase protein.
 12. The methodof claim 11, wherein the agent that binds to a nicotinamide sub-domainof ARTD domain is XAV939{3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}or MN-64 {2-[4-(1-methylethyl)phenyl]-4H-1-benzopyran-4-one}; the agentthat binds to an adenosine sub-domain of a Tankyrase protein is IWR-1[4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolynyl-benzamide], JW55{N-[4-[[[[tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl]methyl]amino]carbonyl]phenyl]-2-purancarboxamide},WIKI42-[3-[[4-(4-methoxyphenyl)-5-(4-pyridynyl)-4H-1,2,4-triazol-3-yl]thio]propyl]-1Hbenz[de]isoquinoline-1,3(2H)-dion,TC-E5001{3-(4-methoxyphenyl)-5-[[[4-(4-methoxyphenyl)-5-methyl-4H-1,2,4-triazol-3-yl]thio]methyl]-1,2,4-oxadiazolor G007-LK{(E)-4-(5-(2-(4-(2-chlorophenyl)-5-(5-(methylsulfonyl)pyridine-2-yl)-4H-1,2,4-triazol-3-yl)vinyl)-1,3,4-oxadiazole-2-yl)benzonitrile};and the agent that binds to an unidentified domain of a Tankyraseprotein is G244-LM {3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazynyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one}, or AZ6102{rel-2-[4-[6-[(3R,5S)-3,5-dimethyl-1-piperazynyl]-4-methyl-3-pyridynyl]phenyl]-3,7-dihydro-7-methyl-4H-pyrrolo[2,3-d]pyrimidine-4-one},or isomers or derivative thereof.
 13. The method of claim 2, wherein theinhibitor is a siRNA that suppresses the expression of Tankyrase geneinto a Tankyrase protein.
 14. The method of claim 13, wherein the siRNAis a dsRNA consisting of RNAs of SEQ ID NO: X1 and SEQ ID NO:X2; or adsRNA consisting of RNAs of SEQ ID NO: X3 and SEQ ID NO:X4.
 15. Themethod of claim
 2. wherein the adult stem cell is autologous orallogenic.
 16. The method of claim
 2. wherein the adult stem cell is amesenchymal stem cell.